Isolation and expression of a gene for a nitrilase from Acidovorax facilis 72W

ABSTRACT

Recombinant microbial strains are provided that express nitrilase enzyme and are useful as biocatalysts for the hydrolysis of nitrile-containing substrates. The recombinant cells are transformed with a foreign gene isolated from  Acidovorax facilis  72W encoding a thermostable nitrilase enzyme that catalyzes the hydrolysis of nitrile-containing substrates to carboxylic acids under mild reaction conditions. The nucleotide sequence of the nitrilase gene and the deduced amino acid sequence encoded by the nitrilase gene are provided.

FIELD OF INVENTION

[0001] The present invention relates to the field of molecular biologyand the use of recombinant microorganisms to express desired genes andgene products. More specifically, the gene used in the invention is anovel nucleic acid fragment encoding a nitrilase enzyme that catalyzesthe hydrolysis of a wide variety of nitrile-containing substances toproduce a corresponding carboxylic acid. Also provided are recombinantstrains that express nitrilase activity and are useful as biocatalystsfor the hydrolysis of nitrile-containing substrates. Additionally, theinvention relates to specific nucleic acids that aid in isolating suchnitrilase genes.

BACKGROUND

[0002] Nitriles are readily converted to the corresponding carboxylicacids by a variety of chemical processes, but these processes typicallyrequire strongly acidic or basic reaction conditions and high reactiontemperatures, and usually produce unwanted byproducts and/or largeamounts of inorganic salts as unwanted waste.

[0003] Processes in which enzyme-catalyzed hydrolysis convertsnitrile-containing substrates to the corresponding carboxylic acids areoften preferred to chemical methods because these processes 1) are oftenrun at ambient temperature, 2) do not require the use of strongly acidicor basic reaction conditions, and 3) do not produce large amounts ofunwanted byproducts. Especially advantageous over chemical hydrolysis,the enzyme-catalyzed hydrolysis of a variety of aliphatic or aromaticdinitriles can be highly regioselective, where only one of the twonitrile groups is hydrolyzed to the corresponding carboxylic acidammonium salt.

[0004] Enzyme-catalyzed hydrolysis of nitrile substrates to thecorresponding carboxylic acids may be accomplished via a one- ortwo-step reaction (Table 1). TABLE 1 Substrates* Product(s) Enzyme TwoStep Reaction 1 RCN + H₂O RC(O)NH₂ nitrile hydratase Reaction 2RC(O)NH₂ + H₂O RC(O)OH + NH₃ amidase One Step Reaction 1 RCN + 2H₂ORC(O)OH + NH₃ nitrilase

[0005] A wide variety of bacterial genera collectively possess a diversespectrum of nitrile hydratase, amidase, or nitrilase activities,including Rhodococcus, Pseudomonas, Alcaligenes, Arthrobacter, Bacillus,Bacteridium, Brevibacterium, Corynebacterium, Agrobacterium,Micrococcus, and Comamonas. Both aqueous suspensions of thesemicroorganisms and the isolated enzymes have been used to convertnitrites to carboxylic acids. Biotechnological use of these enzymes hasbeen recently reviewed by Cowan et al. (Extremophiles (1998) 2:207-216).

[0006] A nitrilase enzyme directly converts a nitrile to thecorresponding carboxylic acid in aqueous solution without theintermediate formation of an amide. The use of nitrilases for thehydrolysis of aromatic nitrites to the corresponding carboxylic acidammonium salts has been known for many years, but it is only recentlythat the use of nitrilases to convert aliphatic nitrites has beenreported. Kobayashi et al. (Tetrahedron (1990) 46:5587-5590; J.Bacteriology (1990) 172:4807-4815) have described an aliphatic nitrilaseisolated from Rhodococcus rhodochrous K22 which catalyzed the hydrolysisof aliphatic nitrites to the corresponding carboxylic acid ammoniumsalts; several aliphatic α,ω-dinitriles were also hydrolyzed. Anitrilase from Comamonas testosteroni has been isolated which canconvert a range of aliphatic α,ω-dinitriles to either the correspondingω-cyanocarboxylic acid ammonium salt or the dicarboxylic acid diammoniumsalt (CA 2,103,616; and Levy-Schil et al., Gene (1995) 161:15-20).

[0007] The nitrilase activity of unimmobilized Acidovorax facilis 72Wcells has been used in a process to prepare five-membered orsix-membered ring lactams from aliphatic α,ω-dinitriles (U.S. Pat. No.5,858,736). In that process, an aliphatic α,ω-dinitrile is firstconverted to an ammonium salt of an ω-cyanocarboxylic acid in aqueoussolution using a catalyst having an aliphatic nitrilase (EC 3.5.5.7)activity. The ammonium salt of the ω-cyanocarboxylic acid is thenconverted directly to the corresponding lactam by hydrogenation inaqueous solution, without isolation of the intermediateω-cyanocarboxylic acid or ω-amino-carboxylic acid. When the aliphaticα,ω-dinitrile is also unsymmetrically substituted at the α-carbon atom,the nitrilase produces the ω-cyanocarboxylic acid ammonium saltresulting from hydrolysis of the ω-nitrile group with greater than 98%regioselectivity, thereby producing only one of the two possible lactamproducts during the subsequent hydrogenation. For example,2-methyl-glutaronitrile (MGN) was hydrolyzed by unimmobilized Acidovoraxfacilis 72W cells to produce 4-cyanopentanoic acid (4-CPA) ammonium saltwith greater than 98% regioselectivity at 100% conversion.

[0008] Nitrilase genes have been cloned and expressed in heterologoussystems, especially in Escherichia coli. Petre et al. (U.S. Pat. No.5,635,391) disclose expression of a nitrilase from Comamonastestosteroni in E. coli and Pseudomonas putida. Also disclosed is amethod to improve levels of soluble nitrilase protein in E. coli bycoexpression of the GroE chaperonin protein, which results in highernitrilase specific activity. The E. coli promoters P_(lac) and P_(trp)were used to drive expression of the nitrilase coding sequences. In E.coli P_(lac) has also been used successfully in expressing nitrilasecoding sequences from Alcaligenes faecalis JM3 (Kobayashi et al., Proc.Nat. Acad. Sci. (1993) 90:247 and JP #4-30663), Rhodococcus rhodochrousJ1 (Kobayashi et al., J. Biol. Chem. (1992) 267:20746) and Rhodococcusrhodochrous K22 (Kobayashi et al., Biochem. (1992) 31, 9000). Stalker(U.S. Pat. No. 4,810,648) discloses that the gene for ahaloarylnitrile-hydrolyzing nitrilase can be expressed under control ofits native promoter in E. coli. In U.S. Pat. No. 5,602,014, Mizumura andYu disclose a specialized regulatory system for expression of nitrilasegenes in Rhodococcus erythropolis.

[0009] Nitrilase enzymes are reported to be highly labile and notobtainable in large quantities (Kobayashi et al., Tetrahedron (1990)46:5587-5590; J. Bacteriology (1990) 172:4807-4815). In contrast tonitrile hydratase, nitrilases are characterized by low specificactivities and reaction rates (Nagasawa et al., Appl Microbiol.Biotechnol. (1993) 40:189-195). The inherent thermal instability ofnitrile-hydrolyzing enzymes from mesophiles is reported to limit theirindustrial applications (Cramp et al., Microbiol. (1997) 143:2313-2320).

[0010] The problem remains the lack of an industrially useful,thermostable, and highly productive nitrilase enzyme suitable as acatalyst for nitrile-containing substrates in applications (such as theregioselective hydrolysis of aliphatic dinitriles to cyanocarboxylicacids) where high yields of product are obtained under mild reactionconditions (including ambient temperatures and without extreme acidic orbasic conditions) and without generating relatively large amounts ofundesirable wastes.

SUMMARY OF THE INVENTION

[0011] The instant invention provides for an isolated DNA sequencespecific to the Acidovorax 72W nitrilase gene. The instant inventionincludes isolated nucleic acid fragments that are complementary to thecomplete sequences of the accompanying Sequence Listing as well assubstantially similar nucleic acid sequences. The instant inventionincludes any nucleic acid fragment that encodes all or a substantialportion of the amino acid sequence of the nitrilase enzyme as set forthin SEQ ID NO:5 or SEQ ID NO:14. The invention also includes an isolatednucleic acid molecule that hybridizes with the nucleic acid fragmentencoding all or a substantial portion of the amino acid sequences of SEQID NO:5 or SEQ ID NO:14 under hybridization conditions of 6×SSC (1MNaCl), 40-45% formamide, 1% SDS at 37° C., and a wash in 0.5× to 1×SSCat 55 to 60° C. Nucleic acid fragments complementary to the listed SEQID NOs:1-16 are also claimed. Polypeptides encoded by the nucleic acidfragments of the invention are also provided, for example, SEQ ID NO:5and SEQ ID NO:14. The invention includes RNA or antisense RNA moleculestranscribed from sequences described above and their cDNA derivatives.

[0012] Also provided are methods to produce active enzyme protein fromthese nitrilase coding sequences in transformed microorganisms. Theimprovement obtained from this invention is an increased reaction rateand higher reactor productivity due to the increased specific activityof the transformant catalysts relative to Acidovorax facilis 72W. Theactive nitrilase enzyme produced in whole cells transformed with geneticmaterials described herein can be used to carry out useful hydrolysis ofnitrile-containing substrates. Transformed microorganisms containingexpression cassettes comprising chimeric genes having plasmidscontaining the isolated nucleic acid fragment encoding nitrilase enzyme,each unit as described herein, are also part of this invention. Oneembodiment uses any of the specific enzyme catalysts E. coli SW91 (ATTCPTA-1175), E. coli DH5α:pnit4 (ATTC PTA-1176), E. coli SS1001 (ATTCPTA-1177), E. coli SS1002 containing plasmid pnitex2, or E. coli SS1011containing plasmid pnitex2 in contact with α,ω-cyanocarboxylic acid. Afurther embodiment uses the specific transformed microorganisms in animproved method to convert α,ω-dinitriles to ω-cyanocarboxylic acids,which are intermediate in the preparation of five-membered orsix-membered ring lactams (See U.S. Pat. No. 5,858,736).

[0013] A method is provided for using a native microbial gene,specifically from Acidorovax facilis 72W, encoding a proteincharacterized by a nitrilase activity on nitrile-containing substratesto obtain a mutated microbial gene encoding a protein characterized byan increased specific nitrilase activity on nitrile-containingsubstrates and/or increased stability of the nitrilase, one or bothcharacteristics increased relative to that of the native microbial gene,the mutated microbial gene produced by a method comprising the steps of

[0014] (i) contacting restriction endonucleases with a mixture ofnucleotide sequences to yield a mixture of restriction fragments, themixture of nucleotide sequences comprising

[0015] a) a native microbial gene;

[0016] b) a first population of nucleotide fragments which willhybridize with the nucleotide sequences of the native microbial gene of(i)(a); and

[0017] c) a second population of nucleotide fragments which will nothybridize to the nucleotide sequences of the native microbial gene of(i)(a),

[0018] (ii) denaturing the mixture of restriction fragments of step (i);

[0019] (iii) incubating the denatured mixture of restriction fragmentsof step (ii) with a polymerase; and

[0020] (iv) repeating steps (ii) and (iii) a sufficient number of timesto yield a mutated microbial gene encoding a protein characterized by anincreased specific nitrilase activity on nitrile-containing substratesand/or increased stability of the nitrilase, on or both characteristicsincreased relative to that of the native microbial gene. The inventionincludes mutated microbial genes produced by this method.

BRIEF DESCRIPTION OF THE FIGURES, BIOLOGICAL DEPOSITS, AND SEQUENCELISTING

[0021] The invention can be more fully understood from the figure, thebiological deposits, the accompanying sequence descriptions, thedetailed description, and claims which together constitute thisapplication.

[0022]FIG. 1 shows a restriction map of the 4.1 kb PstI fragment ofpnit4. The BclI-BglII fragment contains the nitrilase gene and theflanking chromosomal region.

[0023] Applicants have made the following biological deposits under theterms of the Budapest Treaty on the International Recognition of theDeposit of Micro-organisms for the purposes of Patent Procedure:Depositor Identification International Depository Reference DesignationDate of Deposit E. coli SW91 ATCC PTA-1175 11 Jan. 2000 E. coli DH5α:pnit4 ATCC PTA-1176 11 Jan. 2000 E. coli SS1001 ATCC PTA-1177 11 Jan.2000

[0024] As used herein, “ATCC” refers to the American Type CultureCollection International Depository Authority located at ATCC, 10801University Blvd., Manassas, Va. 20110-2209, USA. The “InternationalDepository Designation” is the accession number of the culture ondeposit with ATCC.

[0025] The listed deposit(s) will be maintained in the indicatedinternational depository for at least thirty (30) years and will be madeavailable to the public upon the grant of a patent disclosing it. Theavailability of a deposit does not constitute a license to practice thesubject invention in derogation of patent rights granted by governmentaction.

[0026] Applicant(s) have provided 32 sequences in conformity with 37C.F.R. 1.821-1.825 (“Requirements for Patent Applications ContainingNucleotide Sequences and/or Amino Acid Sequence Disclosures—the SequenceRules”) and consistent with World Intellectual Property Organization(WIPO) Standard ST.25 (1998) and the sequence listing requirements ofthe EPO and PCT (Rules 5.2 and 49.5(a-bis), and Section 208 and Annex Cof the Administrative Instructions). The symbols and format used fornucleotide and amino acid sequence data comply with the rules set forthin 37 C.F.R. §1.822.

[0027] SEQ ID NO:1 is the nucleotide sequence of a forward primer (1F)which was derived from a conserved region found in bacterial nitrilasesequences available from GenBank data and was used as degenerate PCRprimers to discover a novel nitrilase gene.

[0028] SEQ ID NO:2 is the nucleotide sequence of a reverse primer (7R)which was derived from a conserved region found in bacterial nitrilasesequences available from GenBank data and was used as degenerate PCRprimers to discover a novel nitrilase gene.

[0029] SEQ ID NO:3 is the nucleotide sequence of the Acidovorax facilis72W genomic portion of the pJJ28-5 clone without degenerate primerregions.

[0030] SEQ ID NO:4 is the nucleotide sequence of the Acidovorax facilis72W nitrilase coding sequence identified within the 4.1 kb insert frompnit4.

[0031] SEQ ID NO:5 is the amino acid sequence deduced from thenucleotide sequence (SEQ ID NO:4, SEQ ID NO:15) encoded by Acidovoraxfacilis 72W nitrilase coding sequence.

[0032] SEQ ID NO:6 is the nucleotide sequence of a forward primer whichwas used to amplify the nitrilase coding sequence from Acidovoraxfacilis 72W genomic DNA.

[0033] SEQ ID NO:7 is the nucleotide sequence of a reverse primer whichwas used to amplify the nitrilase coding sequence from Acidovoraxfacilis 72W genomic DNA.

[0034] SEQ ID NO:8 is the nucleotide sequence of a forward primer whichwas used to amplify the nitrilase coding sequence from Acidovoraxfacilis 72W genomic DNA.

[0035] SEQ ID NO:9 is the nucleotide sequence of a reverse primer whichwas used to amplify the nitrilase coding sequence from Acidovoraxfacilis 72W genomic DNA.

[0036] SEQ ID NO:10 is the nucleotide sequence of a forward primer whichwas used to amplify the nitrilase coding sequence from Acidovoraxfacilis 72W genomic DNA.

[0037] SEQ ID NO:11 is the nucleotide sequence of a forward primer whichwas used to amplify the nitrilase coding sequence from Acidovoraxfacilis 72W genomic DNA and which was also used to incorporate XhoIrestriction sites.

[0038] SEQ ID NO:12 is the nucleotide sequence of a reverse primer usedto amply the nitrilase coding sequence from Acidovorax facilis 72Wgenomic DNA and which was used to incorporate XhoI restriction sites.

[0039] SEQ ID NO:13 is the nucleotide sequence of the nitrilase codingsequence in plasmid pnitex2 used for overexpression in E. coli.

[0040] SEQ ID NO:14 is the deduced amino acid sequence encoded by thenitrilase coding sequence in plasmid pnitex2 used for overexpression ofnitrilase enzyme in E. coli.

[0041] SEQ ID NO:15 is the nucleotide sequence of a 1776 bp BclI-BglIIgenomic fragment of the plasmid pnit4 containing nitrilase gene andflanking chromosomal region from Acidovorax facilis 72W.

[0042] SEQ ID NO:16 is a synthetic version of the nitrilase gene, withcodon usage optimized for expression in Pichia.

[0043] SEQ ID NOs:17-32 are oligonucleotides used to construct thesynthetic nitrilase gene of SEQ ID NO:16.

DETAILED DESCRIPTION OF THE INVENTION

[0044] Applicants have solved the stated problem with an inventionrelating to the use of a nitrilase gene sequence encoding a proteinuseful as a biocatalyst for the hydrolysis of nitrile-containingsubstrates to a carboxylic acid. The product of nitrile hydrolysis, acarboxylic acid, may also be in the form of a carboxylic acid salt withbyproducts of ammonia or other buffer components. The nitrilase gene wasisolated from Acidovorax facilis 72W. Specifically, recombinant strainsof host cells (such as E. coli) that express active Acidovorax facilis72W nitrilase protein are extremely useful to catalyze the hydrolysis ofa wide variety of nitrile-containing substrates, including the highlyregioselective hydrolysis of dinitriles. Typical dinitriles will havethe formula NC—R—CN where R is an alkylene group having from about 1 toabout 10 carbons.

[0045] This biocatalytic process is industrially attractive as itgenerates much smaller quantities of undesirable wastes and operatesunder much milder conditions than previously known methods. The productsof the present invention are useful as precursors for polymers,solvents, and chemicals of high value in the chemical, agricultural, andpharmaceutical industries.

[0046] Applicants' solution to the problems existing in the technicalfield arose out of the following accomplishments which are discussed inmore detail below and in the Examples:

[0047] I. provided degenerate PCR primer sequences (SEQ ID NOs:1 and 2)useful for identifying the presence of unknown nitrilase genes inbacteria;

[0048] II. isolated nucleic acid sequences useful in screening for thepresence of any nitrilase gene;

[0049] III. mapped, identified, and cloned the complete coding sequencefor a regioselective, thermostable nitrilase from Acidovorax facilis 72W(ATCC 55746) (SEQ ID NO:4 and 15);

[0050] IV. constructed recombinant DNA plasmids pSW91, pnit4, andpnitex2, chimeric genes, and expression cassettes containing the codingsequence as described in III located within a 4.1 kb chromosomal DNAfragment from Acidovorax facilis 72W (ATCC 55746);

[0051] V. constructed recombinant strains of E. coli that express activeAcidovorax facilis 72W nitrilase protein;

[0052] VI. demonstrated regioselective hydrolysis of2-methylglutaronitrile (MGN) to 4-cyanopentanoic acid (4-CPA) usingwhole cells of the recombinant E. coli expressing the Acidovorax facilis72W nitrilase protein described herein; and teach the use of a nativemicrobial gene encoding a protein characterized by a nitrilase activityon nitrile-containing substrates (preferably 2-methylglutaronitrile) toobtain a mutated microbial gene encoding a protein characterized byaltered nitrilase activity and/or greater stability, one or bothcharacteristic increased relative to that of the native nitrilaseprotein.

[0053]Acidovorax facilis 72W nitrilase proved to be an unexpectedlyrobust catalyst for producing carboxylic acids from aliphatic oraromatic nitriles. All known nitrilases, including Acidovorax facilis72W nitrilase, have a nucleophilic cysteine in the enzyme active site(Cowan et al., (1998) supra) and all are susceptible to inactivation bythiol reagents (1.0 mM concentrations of copper chloride, silvernitrate, mercuric acetate, or ferric chloride each produced majordecreases in 72W nitrilase enzyme activity). Cysteine residues are alsocapable of being irreversibly oxidized to sulfinic acids, resulting in aloss of enzyme activity. Despite the sensitivity of nitrilase enzymes tovarious inactivating mechanisms, immobilized Acidovorax facilis 72Wcells used as a catalyst produced up to 3.9×10⁷ moles of product (4-CPA)per mole of nitrilase enzyme (total turnover number, TTN).

[0054] The expression of active Acidovorax facilis 72W nitrilase in aheterologous host cell has several additional advantages over thepreparation and use of Acidovorax facilis 72W cells as a nitrilasecatalyst. The level of expression of the nitrilase protein in Acidovoraxfacilis 72W is ca. 3.4% of total soluble protein (Example 14) and isconstitutive; a thorough screening of potential inducers of nitrilaseproduction (aliphatic or aromatic nitriles or amides, lactams, ureas,etc.) found no effect on the level of nitrilase produced duringfermentation of Acidovorax facilis 72W.

[0055] In contrast, E. coli transformants expressed enzymatically-activeAcidovorax facilis 72W nitrilase at levels of up to 12% of total solubleprotein; the total amount of nitrilase produced (active and inactive) inE. coli transformants was as high as 58% of total soluble protein. Thisincreased level of nitrilase expression in E. coli transformant cellsresults in up to a 2.4-fold increase in specific activity (units ofnitrilase activity per gram dry cell weight) relative to Acidovoraxfacilis 72W cells, which in turn significantly increases the catalystproductivity (g product produced/g dry cell weight/h) (Examples 7 and9). Moreover, Acidovorax facilis 72W cells require glycerol, arelatively expensive carbon substrate, when grown by fermentation, andhave not been successfully grown using inexpensive glucose. In contrast,E. coli transformants can be grown on glucose to the same cell densityas Acidovorax facilis 72W cells in about half the time, significantlyreducing biocatalyst production costs.

[0056]Acidovorax facilis 72W cells additionally contain a nitrilehydratase and an amidase, neither of which are regioselective, and whichcan produce unwanted byproducts when converting α,ω-dinitriles to thecorresponding ω-cyanocarboxylic acid ammonium salts. Acidovorax facilis72W cells required heat-treatment to inactivate the nitrile hydrataseand amidase enzymes (U.S. Pat. No. 5,814,508), risking loss of nitrilaseactivity and adding production costs. Acidovorax facilis mutant strains72-PF-15 and 72-PF-17 were prepared which lacked the nitrile hydrataseenzyme (U.S. Pat. No. 5,858,736), but these strains had only ca. halfthe nitrilase specific activity of the parent Acidovorax facilis 72Wstrain.

[0057] In contrast, an E. coli transformed with the genetic material toexpress nitrilase enzyme does not require a heat-treatment step toinactivate unwanted nitrile hydratase or amidase activities, eliminatingthe cost of this processing step for catalyst production and avoidingthe inadvertent loss of nitrilase activity. E. coli transformants havinga high specific nitrilase activity with the attendant advantages havebeen produced by Applicants as described in Examples 6 and 7.

[0058] The whole-cell nitrilase activity of Acidovorax facilis 72W isvery stable at temperatures of up to about 55° C.; a cell suspension in0.10 M phosphate buffer (pH 7.0) had a nitrilase half-life of 22.7 h at50° C. (Gavagan et al., Appl. Microbiol. Biotechnol. (1999) 52:654-659).The purified enzyme also had excellent temperature stability, where noloss of activity of a 10 mg/mL solution of purified nitrilase in 0.10 Mphosphate buffer was observed after 24 h at 45° C. When stored as asolution in 50 mM potassium phosphate buffer (pH 7.0) at 5° C., no lossof activity of purified nitrilase was observed after 46 days. AlthoughAcidovorax facilis 72W, a mesophilic bacterium, has an optimaltemperature for growth of 32° C., the 72W nitrilase enzyme itself has athermal stability that compares favorably to the nitrilase enzyme of athermophilic bacterium such as the Bacillus pallidus strain DAC521(Cramp et al., Microbiol. (1997) 13:2313-2320).

[0059] When unimmobilized cells are used as a catalyst for hydrolysis ofMGN to 4-CPA, either centrifugation or ultrafiltration was required torecover the unimmobilized cells for reuse. At high productconcentrations (up to 29 wt. % 4-CPA ammonium salt), the unimmobilizedcells lost significant activity with each reuse and cell lysis wasobserved. In contrast, immobilizing the cells simplified catalystrecovery and reuse, improved the resistance of cells to lysis, andincreased the stability of the enzyme activity of the immobilized cellswhen recycled in consecutive batch reactions as compared to usingunimmobilized cells.

[0060] Immobilization of either whole cell Acidovorax facilis 72W or theE. coli transformant SS1100 in carrageenan under identical conditionsproduced gels that were stable when recycled in consecutive batchreactions which produced high concentrations of 4-CPA ammonium salt (upto ca. 200 g/L). Gel beads were prepared by first dispersing a heatedaqueous suspension of cells (5% dry cell weight) and carrageenan (3 wt%) in heated soybean oil at 50° C., and the resulting droplets weregelled by lowering the temperature of the oil below the gellingtemperature of the carrageenan (Audet et al., Process Biochem. (1989)24:217). The cell/carrageenan beads, which had an average diameter offrom 0.5 mm-3 mm, were separated from the soybean oil, then washed withaqueous buffer and crosslinked with glutaraldehyde andpolyethyleneimine.

[0061]Acidovorax facilis 72W cells and the E. coli transformant SS1001cells immobilized in carrageenan beads were compared in side-by-sidereactions under identical conditions for the production of 1.25 M 4-CPAammonium salt. The reaction rate when using immobilized E. colitransformant SS1001 was 1.7 times greater than when using immobilizedAcidovorax facilis 72W cells (310 mM 4-CPA ammonium salt/h and 184 mM4-CPA ammonium salt/h, respectively; Example 9). The increase inreaction rate obtained with E. coli transformant SS1001 was a result ofthe higher specific activity (units of nitrilase activity per gram ofbeads) of the immobilized transformant cell catalyst, and demonstratesone advantage of using a transformant cell with higher specificnitrilase activity relative to the parent Acidovorax facilis 72W strain.The reaction rate when using E. coli transformant SW91 cells immobilizedin calcium alginate beads according to a published procedure (Bucke,Methods Enzymol. (1987) 135:175-189) was 4.0 times greater than whenusing carrageenan-immobilized Acidovorax facilis 72W cells (739 mM 4-CPAammonium salt/h and 184 mM 4-CPA ammonium salt/h, respectively; Examples9 and 11). The increased catalyst productivity (g product/g catalyst/h)achieved by using the immobilized transformant catalyst significantlyreduces process costs.

[0062] The invention provides a new nucleic acid sequence encodingnitrilase enzyme. This sequence comprises an open reading frame (ORF)residing on a 4.1 kb PstI fragment isolated from Acidovorax facilis 72Wgenomic DNA. The newly defined ORF encodes an identifiable enzyme thatconverts nitrile to the corresponding carboxylic acid ammonium salt. TheORF was identified both on the basis of expression of active nitrilaseas well as comparison of the nucleic acid and deduced amino acidsequences to public databases using algorithms well known in the art.The protein coded for by the ORF showed up to a 71% primary amino acidsequence identity to other known nitrilases.

[0063] Accordingly, preferred polypeptides of the instant invention arethose active proteins that are at least 80% identical to the amino acidsequence reported herein. More preferred amino acid fragments are atleast 90% identical to the sequences herein. Most preferred are aminoacid fragments that are at least 95% identical to the amino acidfragments reported herein. Similarly, preferred nucleic acid sequencescorresponding to the instant ORF are those encoding active proteins andwhich are at least 80% identical to the nucleic acid sequences reportedherein. More preferred nucleic acid fragments are at least 90% identicalto the sequences herein. Most preferred are nucleic acid fragments thatare at least 95% identical to the nucleic acid fragments reportedherein.

[0064] The nucleic acid fragments of the instant invention may be usedto isolate cDNAs and genes encoding homologous enzymes from the same orother bacterial species. Isolation of homologous genes usingsequence-dependent protocols is well known in the art. Examples ofsequence-dependent protocols include, but are not limited to, methods ofnucleic acid hybridization, and methods of DNA and RNA amplification asexemplified by various uses of nucleic acid amplification technologies(e.g., polymerase chain reaction, ligase chain reaction).

[0065] In general, a sequence of ten or more contiguous amino acids orthirty or more nucleotides is necessary to putatively identify apolypeptide or nucleic acid sequence as homologous to a known protein orgene. Moreover, with respect to nucleotide sequences, gene-specificoligonucleotide probes comprising 20-30 contiguous nucleotides may beused in sequence-dependent methods of gene identification (e.g.,Southern hybridization) and isolation (e.g., in situ hybridization ofbacterial colonies or bacteriophage plaques). In addition, shortoligonucleotides of 12-15 bases may be used as amplification primers inPCR in order to obtain a particular nucleic acid fragment comprising theprimers.

[0066] For example, genes encoding similar enzymes to that of theinstant invention, either as cDNAs or genomic DNAs, could be isolateddirectly by using all or a portion of the instant nucleic acid fragmentsas DNA hybridization probes to screen libraries from any desiredbacteria using methodology well known to those skilled in the art.Specific oligonucleotide probes based upon the instant nucleic acidsequences can be designed and synthesized by methods known in the art(Sambrook, J., Fritsch, E. F. and Maniatis, T., Molecular Cloning: ALaboratory Manual (1989), Cold Spring Harbor Laboratory Press, ColdSpring Harbor (referred to throughout as “Maniatis”). Moreover, theentire sequences can be used directly to synthesize DNA probes bymethods known to the skilled artisan such as random primers DNAlabeling, nick translation, or end-labeling techniques, or RNA probesusing available in vitro transcription systems. In addition, specificprimers can be designed and used to amplify a part of or full-length ofthe instant sequences. The resulting amplification products can belabeled directly during amplification reactions or labeled afteramplification reactions, and used as probes to isolate full length cDNAor genomic fragments under conditions of appropriate stringency.

[0067] Two short segments of the instant ORF may be used in polymerasechain reaction protocols to amplify longer nucleic acid fragmentsencoding homologous genes from DNA or RNA. Alternatively, the secondprimer sequence may be based upon sequences derived from the cloningvector. For example, the skilled artisan can follow the RACE protocol togenerate cDNAs by using PCR to amplify copies of the region between asingle point in the transcript and the 3′ or 5′ end. Primers oriented inthe 3′ and 5′ directions can be designed from the instant sequences.Using commercially available 3′ RACE or 5′ RACE systems (BRL), specific3′ or 5′ cDNA fragments can be isolated (Ohara et al., PNAS USA (1989)86:5673; Loh et al., Science (1989) 243:217).

[0068] Typically, in PCR-type amplification techniques, the primers havedifferent sequences and are not complementary to each other. Dependingon the desired test conditions, the sequences of the primers should bedesigned to provide for both efficient and faithful replication of thetarget nucleic acid. Methods of PCR primer design are common and wellknown in the art. (Thein and Wallace, “The use of oligonucleotide asspecific hybridization probes in the diagnosis of genetic disorders”(1986) pp. 33-50, in Human Genetic Diseases: A Practical Approach, K. E.Davis (Ed.), IRL Press, Herndon, Va.); Rychlik, W., PCR Protocols:Current Methods and Applications. (1993) 15:31-39, in Methods inMolecular Biology, B. A. White, (Ed.), Humania Press, Inc., Totowa,N.J.) Alternatively, the instant sequences may be used as hybridizationreagents for the identification of homologs. The basic components of anucleic acid hybridization test include a probe, a sample suspected ofcontaining the gene or gene fragment of interest, and a specifichybridization method. Probes of the present invention are typicallysingle stranded nucleic acid sequences which are complementary to thenucleic acid sequences to be detected. Probes are “hybridizable” to thenucleic acid sequence to be detected (cDNA, genomic DNA or RNA). Theprobe length can vary from 5 bases to tens of thousands of bases, thelength depending upon the specific test to be done. Only part of theprobe molecule need be complementary to the nucleic acid sequence to bedetected. In addition, the complementarity between the probe and thetarget sequence need not be perfect. Hybridization does occur betweenimperfectly complementary molecules with the result that a certainfraction of the bases in the hybridized region are not paired with theproper complementary base.

[0069] Hybridization methods are well defined (Maniatis, particularlyChapter 11 and Table 11.1). Typically, the probe and sample must bemixed under conditions which will permit nucleic acid hybridization.This involves contacting the probe and sample in the presence of aninorganic or organic salt under appropriate temperature and ionicstrength conditions. The probe and sample nucleic acids must be incontact for a long enough time that any possible hybridization betweenthe probe and sample nucleic acid may occur. The concentration of probeor target in the mixture will determine the time necessary forhybridization to occur. The greater the probe or target concentration,the shorter the hybridization incubation time needed. Optionally, achaotropic agent may be added. The chaotropic agent stabilizes nucleicacids by inhibiting nuclease activity. Furthermore, the chaotropic agentallows sensitive and stringent hybridization of short oligonucleotideprobes at room temperature (Van Ness et al., Nucl. Acids Res. (1991)19:5143-5151). Suitable chaotropic agents include guanidinium chloride,guanidinium thiocyanate, sodium thiocyanate, lithium tetrachloroacetate,sodium perchlorate, rubidium tetrachloroacetate, potassium iodide, andcesium trifluoroacetate, among others. Typically, the chaotropic agentwill be present at a final concentration of about 3M. If desired, onecan add formamide to the hybridization mixture, typically 30-50% (v/v).

[0070] Various hybridization solutions can be used. Typically, thesecomprise from about 20 to 60% volume, preferably 30%, of a polar organicsolvent. A common hybridization solution employs about 30-50% v/vformamide, about 0.15 to 1M sodium chloride, about 0.05 to 0.1M buffers,such as sodium citrate, Tris-HCl, PIPES or HEPES (pH range about 6-9),about 0.05 to 0.2% detergent, such as sodium dodecylsulfate, or between0.5-20 mM EDTA, FICOLL (Pharmacia Inc.) (about 300-500 kilodaltons),polyvinylpyrrolidone (about 250-500 kilodaltons), and serum albumin.Also included in the typical hybridization solution will be unlabeledcarrier nucleic acids from about 0.1 to 5 mg/mL, fragmented nucleic DNA,e.g., calf thymus or salmon sperm DNA, or yeast RNA, and optionally fromabout 0.5 to 2% wt./vol. glycine. Other additives may also be included,such as volume exclusion agents which include a variety of polarwater-soluble or swellable agents, such as polyethylene glycol, anionicpolymers such as polyacrylate or polymethylacrylate, and anionicsaccharidic polymers, such as dextran sulfate.

[0071] Nucleic acid hybridization is adaptable to a variety of assayformats. One of the most suitable is the sandwich assay format. Thesandwich assay is particularly adaptable to hybridization undernon-denaturing conditions. A primary component of a sandwich-type assayis a solid support. The solid support has adsorbed to it or covalentlycoupled to it immobilized nucleic acid probe that is unlabeled andcomplementary to one portion of the sequence.

[0072] Availability of the instant nucleotide and deduced amino acidsequences facilitates immunological screening of cDNA expressionlibraries. Synthetic peptides representing portions of the instant aminoacid sequences may be synthesized. These peptides can be used toimmunize animals to produce polyclonal or monoclonal antibodies withspecificity for peptides or proteins comprising the amino acidsequences. These antibodies can be then be used to screen cDNAexpression libraries to isolate full-length cDNA clones of interest(Lerner, Adv. Immunol. 36:1 (1984); and Maniatis).

[0073] Heterologous Host Cells:

[0074] The active Acidovorax facilis 72W nitrilase protein may beproduced in heterologous host cells, preferably in microbial hosts.Particularly useful in the present invention will be cells that arereadily adaptable to large-scale fermentation methods. Such organismsare well known in the art of industrial bioprocessing, examples of whichmay be found in “Recombinant Microbes for Industrial and AgriculturalApplications”, Murooka et al., eds., Marcel Dekker, Inc., New York, N.Y.(1994), and include fermentative bacteria as well as yeast andfilamentous fungi. Host cells may include but are not limited toComamonas sp., Corynebacterium sp., Brevibacterium sp., Rhodococcus sp.,Azotobacter sp., Citrobacter sp., Enterobacter sp., Clostridium sp.,Klebsiella sp., Salmonella sp., Lactobacillus sp., Aspergillus sp.,Saccharomyces sp., Zygosaccharomyces sp., Pichia sp., Kluyveromyces sp.,Candida sp., Hansenula sp., Dunaliella sp., Debaryomyces sp., Mucor sp.,Torulopsis sp., Methylobacteria sp., Bacillus sp., Escherichia sp.,Pseudomonas sp., Rhizobium sp., and Streptomyces sp. Particularlypreferred is E. coli. Examples of suitable E. coli host cells in which anitrilase gene can be expressed include but are not limited to hostcells specified herein and MG1655 (ATCC 47076), W3110 (ATCC 27325),MC4100 (ATCC 35695), W1485 (ATCC 12435), and their derivatives.

[0075] Microbial Expression Systems and Expression Vectors:

[0076] Microbial expression systems and expression vectors containingregulatory sequences that direct high level expression of foreignproteins are well known to those skilled in the art. These could be usedto construct chimeric genes for production of the gene products of the4.1 kb fragment of the invention. These chimeric genes could then beintroduced into appropriate microorganisms via transformation to providehigh level expression of the nitrilase enzymes. The nucleotides of thepresent invention may be used to produce gene products having enhancedor altered activity levels relative to that of the native gene sequence.

[0077] Additionally, chimeric genes will be effective in altering theproperties of a host cell. For example, introducing at least one copy ofchimeric genes encoding the present ORF under the control of theappropriate promoters into a host cell gives the host cell the abilityto convert 2-methylglutaronitrile to 4-cyanopentanoic acid. The chimericgenes of the instant invention will comprise suitable regulatorysequences useful for driving gene expression of the present nitrilasesequences. Regulatory sequences will include, but are not limited topromoters, translation leader sequences, and ribosomal binding sites. Itis preferred if these sequences are derived from the host organism;however, the skilled person will recognize that heterologous regulatorysequences may also be used.

[0078] In one embodiment, the regulatory sequences will include apromoter. Promoters may be constitutive or inducible. Induciblepromoters are generally responsive to a specific stimulus (e.g., IPTGinducing the lac promoter). Inducible promoters may be responsive to avariety of stimuli, including, chemicals, growth cycle, changes intemperature, changes in pH and changes in osmolarity, to name only afew. In a preferred embodiment of the invention it has been discoveredthat a chimeric gene comprising a nitrilase coding regions (e.g., SEQ IDNO:4, SEQ ID NO:130R SEQ ID NO:15) is expressed as effectively, or moreeffectively in the absence of an inducer where the regulatory regionscomprise an inducible promoter.

[0079] The chimeric gene is introduced into the appropriate host bycloning it into a suitable expression vector. Vectors or cassettesuseful for the transformation of suitable host cells are well known inthe art. Typically, the vector or cassette contains sequences directingtranscription and translation of the relevant gene, a selectable marker,and sequences allowing autonomous replication or chromosomalintegration. Suitable vectors comprise a region 5′ of the codingsequence that harbors transcriptional initiation controls and a region3′ of the DNA fragment which controls transcriptional termination. It ismost preferred when both control regions are derived from geneshomologous to the host cell, although such control regions need not bederived from the genes native to the specific species chosen as aproduction host.

[0080] Initiation control regions or promoters, which are useful todrive expression of the instant ORF in the desired host cell arenumerous and familiar to those skilled in the art, including but notlimited to CYC1, HIS3, GAL1, GAL10, ADH1, PGK, PHO5, GAPDH, ADC1, TRP1,URA3, LEU2, ENO, TPI (useful for expression in Saccharomyces); AOX1(useful for expression in Pichia); and lac, trp, 1P_(L), 1P_(R), T7,tac, P_(BAD), and trc (useful for expression in Escherichia coli).Examples include at least one of the promoters selected from the groupsconsisting of the tryptophan operon promoter Ptrp of E. coli, a lactoseoperon promoter Plac of E. coli, a Ptac promoter of E. coli, a phagelambda right promoter PR, a phage lambda left promoter PL, a T7promoter, and a promoter of the GAP gene from Pichia pastoris, or is atleast one strong promoter selected from the group of microorganismsconsisting of Comamonas, Corynebacterium, Brevibacterium, Rhodococcus,Azotobacter, Citrobacter, Enterobacter, Clostridium, Klebsiella,Salmonella, Lactobacillus, Aspergillus, Saccharomyces, Pichia,Zygosaccharomyces, Kluyveromyces, Candida, Hansenula, Dunaliella,Debaryomyces, Mucor, Torulopsis, Methylobacteria, Bacillus, Escherichia,Pseudomonas, Rhizobium, and Streptomyces.

[0081] Termination control regions may also be derived from variousgenes native to the preferred hosts. Optionally, a termination site maybe unnecessary; however, it is most preferred if included.

[0082] Additionally, the inserted genetic material may include aribosome binding site. The ribosome binding site may be from a phagelambda CII gene or is selected from the group consisting of ribosomebinding sites from a gene of Comamonas, Corynebacterium, Brevibacterium,Rhodococcus, Azotobacter, Citrobacter, Enterobacter, Clostridium,Klebsiella, Salmonella, Lactobacillus, Aspergillus, Saccharomyces,Zygosaccharomyces, Pichia, Kluyveromyces, Candida, Hansenula,Dunaliella, Debaryomyces, Mucor, Torulopsis, Methylobacteria, Bacillus,Escherichia, Pseudomonas, Rhizobium, and Streptomyces.

[0083] Optionally, the instant gene product may preferably be asecretion product of the transformed host. Secretion of desired proteinsinto the growth media simplifies purification procedures and reducescosts. Secretion signal sequences are often useful in facilitating theactive transport of expressible proteins across cell membranes. Atransformed host capable of secretion may be created by incorporating inthe host a DNA sequence that codes for a secretion signal. Methods forchoosing appropriate signal sequences are well known in the art (see forexample EP 546049; WO 9324631). The secretion signal DNA may be locatedbetween the expression-controlling DNA and the instant coding sequenceor coding sequence fragment, and in reading frame with the latter.

[0084] Fermentations, which may be run in the batch, fed-batch, orcontinuous mode, are common and well known in the art (Thomas D. Brockin Biotechnology: A Textbook of Industrial Microbiology, Second Edition(1989) Sinauer Associates, Inc.; Sunderland et al., Appl. Biochem.Biotechnol. (1992) 36:227.

[0085] The present nucleotide fragments may be used to produce geneproducts having enhanced or altered activity levels. Specifically, thenucleotide fragments can be used to produce a mutated microbial geneencoding a protein characterized by an increased nitrilase activity onnitrile-containing substrates and or an increased enzyme stability, oneor both characteristics increased relative to the nitrilase activity ofthe native microbial gene. Various methods are known for mutating anative gene sequence to produce a gene product with altered or increasedactivity relative to the native gene sequence. These include, but arenot limited to, directed evolution, random mutagenesis, domain swapping(using zinc finger domains, or restriction enzymes), rational design,error prone PCR (Melnikov et al., Nucleic Acids Research (1999)27(4):1056-1062); site-directed mutagenesis (Coombs et al., in Proteins(1998) 259-311, R. H. Angeletti (Ed.), Academic Press, San Diego,Calif.), and “gene shuffling” (U.S. Pat. No. 5,605,793; U.S. Pat. No.5,811,238; U.S. Pat. No. 5,830,721; and U.S. Pat. No. 5,837,458,incorporated herein by reference).

[0086] Definitions of Abbreviations and Terms:

[0087] In this specification, a number of terms and abbreviations areused in the manner defined below.

[0088] The abbreviations in the specification correspond to units ofmeasure, techniques, properties, or compounds as follows: “sec” meanssecond(s), “min” means minute(s), “h” means hour(s), “d” means day(s),“μL” means microliter, “mL” means milliliters, “L” means liters, “mM”means millimolar, “M” means molar, “mmol” means millimole(s), “Ampr”means ampicillin resistance, “Amps” means ampicillin sensitivity, “kb”means kilo base, “kd” means kilodaltons, “nm” means nanometers, and “wt”means weight. “ORF” means open reading frame, “PCR” means polymerasechain reaction, “SSC” means saline-sodium citrate buffer, “HPLC” meanshigh performance liquid chromatography, “ca” means approximately, “dcw”means dry cell weight, “O.D.” means optical density at the designatedwavelength, “IU” means International Units, “MGN” means2-methylglutaronitrile, “4-CPA” means 4-cyanopentanoic acid, and “IPTG”means isopropyl β-D-thiogalactopyranoside.

[0089] “Enzyme catalyst” refers to a catalyst characterized by aspecific nitrilase activity on nitrile-containing substrates.

[0090] “Hydrogenation catalyst” refers to a material that accelerateshydrogenation without itself being consumed or undergoing a chemicalchange. Hydrogenation catalysts suitable for use in this inventioninclude, but are not limited to, the various platinum metals, such asiridium, osmium, rhodium, ruthenium, platinum, and palladium; alsovarious other transition metals such as cobalt, copper, nickel and zinc.The catalyst may be unsupported, (for example, as Raney nickel orplatinum oxide), or it may be supported (for example, as palladium oncarbon, platinum on alumina, or nickel on kieselguhr).

[0091] The terms “host cell” and “host organism” refer to a cell capableof receiving foreign or heterologous genes, gene fragments, or DNAfragments.

[0092] The term “mesophilic baterium” refers to a bacterium living inthe temperature range near that of warm-blooded animals, and usuallyshowing a growth temperature optimum between 25 and 40° C.

[0093] The terms “recombinant organism”, “transformed host”,“transformant”, “transgenic organism”, and “transformed microbial host”refer to a host organism having been transformed with heterologous orforeign DNA. The recombinant organisms of the present invention expressforeign coding sequences or genes which encode active nitrilase enzyme.“Transformation” refers to the transfer of a DNA fragment into thegenome of a host organism, resulting in genetically stable inheritance.“Transformation cassette” refers to a specific fragment of DNAcontaining a set of genetic elements conveniently arranged for insertioninto a host cell, usually as part of a plasmid. “Expression cassette”refers to a specific fragment of DNA containing a set of geneticelements conveniently arranged for insertion into a host cell, usuallyas part of a plasmid, that also allows for enhanced gene expression inthe host.

[0094] The terms “plasmid” and “vector” refer to an extra chromosomalelement often carrying genes which are not part of the centralmetabolism of the host cell, and usually in the form of circulardouble-stranded DNA molecules. Such elements may be autonomouslyreplicating sequences, genome integrating sequences, phage or nucleotidesequences, linear or circular, of a single- or double-stranded DNA orRNA, derived from any source, in which a number of nucleotide sequenceshave been joined or recombined into a unique construction.

[0095] The term “nucleic acid” refers to complex compounds of highmolecular weight occurring in living cells, the fundamental units ofwhich are nucleotides linked together with phosphate bridges. Nucleicacids are subdivided into two types: ribonucleic acid (RNA) anddeoxyribonucleic acid (DNA).

[0096] The letters “A”, “G”, “T”, and “C” when referred to in thecontext of nucleic acids, mean the purine bases (Adenine (C₅H₅N₅) andGuanine (C₅H₅N₅O)) and the pyrimidine bases (Thymine (C₅H₆N₂O₂) andCytosine (C₄H₅N₃O)), respectively.

[0097] The term “complementary” is used to describe the relationshipbetween nucleotide bases that are hybridizable to one another. Forexample, with respect to DNA, adenosine is complementary to thymine andcytosine is complementary to guanine. cDNA is a single stranded DNAcomplementary to an RNA synthesized from it by reverse transcription invitro. Anti-sense RNA is an RNA molecule complementary to another RNA.

[0098] The terms “coding sequence” or “coding region” refers to a DNAsequence that codes for a specific amino acid sequence. The terms “ORF”and “open reading frame” and “coding sequence” and “coding region” areused interchangeably to refer to a portion of DNA sequence thattranslates into a protein. ORFs are usually delineated in the sequenceby three base pairs designating the start (a start codon) and three basepairs designating the stop (a stop codon) in the translation of the DNAsequence into the protein sequence.

[0099] The terms “nucleic acid fragment” or “nucleotide fragment” referto a fragment of DNA that may encode a gene and/or regulatory sequencespreceding (5′, upstream) or following (3′, downstream) the codingsequence. A “fragment” constitutes a fraction of the complete nucleicacid sequence of a particular region. A fragment may constitute anentire gene.

[0100] The terms “restriction endonuclease” and “restriction enzyme”refer to an enzyme which catalyzes hydrolytic cleavage within a specificnucleotide sequence in double-stranded DNA.

[0101] The term “oligonucleotide” refers to primers, probes, oligomerfragments to be detected, labeled-replication blocking probes, andoligomer controls, and refers generically to polydeoxyribonucleotides(containing 2-deoxy-D-ribose), to polyribonucleotides (containingD-ribose) and to any polynucleotide which is an N-glycoside of a purineor pyrimidine base (nucleotide), or modified purine or pyrimidine base.Also included in the definition of “oligonucleotide” are nucleic acidanalogs (e.g., peptide nucleic acids) and those that have beenstructurally modified (e.g., phosphorothiolate linkages) (See alsoThuong et al., Biochimie (1985) Jul-Aug 67(7-8):673-684.) There is nointended distinction between the length of a “nucleic acid”,“polynucleotide” or an “oligonucleotide”.

[0102] The term “primer” refers to an oligonucleotide (synthetic oroccurring naturally), which acts as a point of initiation of nucleicacid synthesis or replication along a complementary strand when placedunder conditions in which synthesis of a complementary strand iscatalyzed by a polymerase.

[0103] The term “probe” refers to an oligonucleotide (synthetic oroccurring naturally), that is significantly complementary to a“fragment” and forms a duplexed structure by hybridization with at leastone strand of the fragment.

[0104] “Suitable regulatory sequences” refer to nucleotide sequenceswhich influence the transcription, RNA processing, RNA stability, ortranslation of the associated coding sequence and which are locatedupstream (5′ noncoding sequences), within, or downstream (3′ noncodingsequences) of a coding sequence. Regulatory sequences may includepromoters, translation leader sequences, introns, and polyadenylationrecognition sequences.

[0105] “Promoter” refers to a DNA sequence capable of controlling theexpression of a coding sequence or functional RNA. In general, a codingsequence is located 3′ to a promoter sequence. Promoters may be derivedin their entirety from a native gene, or be composed of differentelements derived from different promoters found in nature, or evencomprise synthetic DNA segments. It is understood by those skilled inthe art that different promoters may direct the expression of a gene indifferent tissues or cell types, or at different stages of development,or in response to different environmental conditions. Promoters thatcause a gene to be expressed in most cell types at most times or undermost environmental conditions are commonly referred to as “constitutivepromoters”. Promoters that cause a gene to be expressed only in thepresence of a particular compound or environmental condition arecommonly referred to as “inducible promoters”. Since in most cases theexact boundaries of regulatory sequences have not been completelydefined, DNA fragments of different lengths may have identical promoteractivity.

[0106] The term “operably linked” refers to the association of nucleicacid sequences on a single nucleic acid fragment so that the function ofone is affected by the other. For example, a promoter is operably linkedwith a coding sequence when it is capable of affecting the expression ofthat coding sequence (i.e., that the coding sequence is under thetranscriptional control of the promoter). Coding sequences can beoperably linked to regulatory sequences in sense or antisenseorientation.

[0107] The “3′ non-coding sequences” refer to DNA sequences locateddownstream of a coding sequence and include polyadenylation recognitionsequences and other sequences encoding regulatory signals capable ofaffecting mRNA processing or gene expression. The polyadenylation signalis usually characterized by affecting the addition of polyadenylic acidtracts to the 3′ end of the mRNA precursor.

[0108] “Gene” refers to a nucleic acid fragment that expresses aspecific protein, including regulatory sequences preceding (5′non-coding sequences) and following (3′ non-coding sequences) the codingsequence. “Native gene” refers to a gene with its own regulatorysequences in an arrangement as found in nature. “Chimeric gene” refersto any gene that is not a native gene, comprising regulatory and codingsequences that are not found together in nature. Accordingly, a chimericgene may comprise regulatory sequences and coding sequences that arederived from different sources, or regulatory sequences and codingsequences derived from the same source, but arranged in a mannerdifferent than that found in nature. “Endogenous gene” refers to anative gene in its natural location in the genome of the nativeorganism. A “foreign” gene refers to a gene not normally found in thehost organism, but that is introduced into the host organism by genetransfer. Foreign genes can comprise native genes inserted into anon-native organism, or chimeric genes. A “transgene” is a gene that hasbeen introduced into the genome by a transformation procedure.

[0109] “Synthetic genes” can be assembled from oligonucleotide buildingblocks that are chemically synthesized using procedures known to thoseskilled in the art. These building blocks are ligated and annealed toform gene segments that are then enzymatically assembled to constructthe entire gene. “Chemically synthesized”, as related to a sequence ofDNA, means that the component nucleotides were assembled in vitro.Manual chemical synthesis of DNA may be accomplished usingwell-established procedures, or automated chemical synthesis can beperformed using one of a number of commercially available machines.

[0110] The skilled artisan is well aware of the “codon-bias” exhibitedby a specific host cell in using nucleotide codons to specify a givenamino acid. Therefore, when synthesizing a gene for improved expressionin a host cell, it is desirable to design the gene such that its codonusage reflects the preferred codon bias of the host cell. A survey ofgenes derived from the host cell where sequence information is availablecan determine its codon bias.

[0111] The term “expression” means the transcription and translation togene product from a gene coding for the sequence of the gene product,usually a protein.

[0112] The terms “protein”, “polypeptide”, and “peptide” are usedinterchangeably to refer to the gene product expressed. “Mature” proteinrefers to a post-translationally processed polypeptide (i.e., one fromwhich any pre- or pro-peptides present in the primary translationproduct have been removed). “Precursor” protein refers to the primaryproduct of translation of mRNA (i.e., with pre- and pro-peptides stillpresent). Pre- and pro-peptides may be, but are not limited to,intracellular localization signals.

[0113] The term “percent identity” is a relationship between two or morepolypeptide sequences or two or more polynucleotide sequences, asdetermined by comparing the sequences. “Identity” also means the degreeof sequence relatedness between polypeptide or polynucleotide sequences,as the case may be, as determined by the match between strings of suchsequences.

[0114] “Similarity” is a descriptive term that only implies that twosequences, by some criterion, resemble each other and carries nosuggestion as to their origins or ancestor. “Homology” refersspecifically to similarity due to descent from a common ancestor. On thebasis of similarity relationships among a group of sequences, it may bepossible to infer homology, but outside of an explicit laboratory modelsystem, descent from a common ancestor remains hypothetical. (States etal., Similarity and Homogeneity (1992) pp. 89-92, in J. SequenceAnalysis Primer, Gribskov, M. and Devereux, J., (Eds.), Freeman andCo.).

[0115] “Identity” and “similarity” can be readily calculated by knownmethods including, but not limited to, those described in ComputationalMolecular Biology (Lesk, A. M., ed.) Oxford University Press, New York(1988); Biocomputing: Informatics and Genome Projects (Smith, D. W.,ed.) Academic Press, New York (1993); Computer Analysis of SequenceData, Part I (Griffin, A. M., and Griffin, H. G., eds.) Humana Press,Totowa, N.J. (1994); Sequence Analysis in Molecular Biology (von Heinje,G., ed.) Academic Press (1987); and Sequence Analysis Primer (Gribskov,M. and Devereux, J., Eds.) Stockton Press, New York (1991). Preferredmethods to determine identity are designed to give the best matchbetween the sequences tested.

[0116] Preferred computer program methods to determine the identity andsimilarity between two sequences include, but are not limited to, theWisconsin Package Version 9.0 and 10.0 Genetics Computer Group (GCG) Gapprogram found in the GCG program package, BLASTP, BLASTN, and FASTA(Pearson et al., Proc. Natl. Acad. Sci. USA. (1988) 85:2444-2448). TheBLAST X program is publicly available from NCBI and other sources (BLASTManual, Altschul et al., Natl. Cent. Biotechnol. Inf., Natl. LibraryMed. (NCBI NLM) NIH, Bethesda, Md. 20894; Altschul et al., J. Mol. Biol.(1990) 215:403-410).

[0117] The term “substantially similar” refers to nucleic acid fragmentswherein changes in one or more nucleotide bases results in substitutionof one or more amino acids, but do not affect the functional propertiesof the protein encoded by the DNA sequence. “Substantially similar” alsorefers to nucleic acid fragments wherein changes in one or morenucleotide bases does not affect the ability of the nucleic acidfragment to mediate alteration of gene expression by antisense orco-suppression technology. “Substantially similar” also refers tomodifications of the nucleic acid fragments of the instant inventionsuch as substitution, deletion or insertion of one or more nucleotidebases that do not substantially affect the functional properties of theresulting transcript.

[0118] “Codon degeneracy” refers to the divergence in the genetic codepermitting variation of the nucleotide sequence without affecting theamino acid sequence of an encoded polypeptide. For example, it is wellknown in the art that the triplet codons CTT, CTC, CTA, and CTG all codefor the amino acid leucine (Atlas, R., Principles of Microbiology). Itis also well known in the art that alterations in a gene that produce achemically-equivalent amino acid at a given site, but do not affect thefunctional properties of the encoded protein, are common. Thus, a codonfor the amino acid alanine, a hydrophobic amino acid, may be substitutedby a codon encoding another less hydrophobic residue (such as glycine)or a more hydrophobic residue (such as valine, leucine, or isoleucine)without affecting the functional properties of the encoded protein.Similarly, substitution of one negatively charged residue for another(such as aspartic acid for glutamic acid) or one positively chargedresidue for another (such as lysine for arginine) can also be expectedto produce a functionally equivalent product. Nucleotide changes thatresult in alteration of the N-terminal and C-terminal portions of theprotein molecule would also not be expected to alter the activity of theprotein. Each of the proposed modifications is well within the routineskill in the art, as is determining if the biological activity of theencoded products is retained.

[0119] Moreover, the skilled artisan recognizes that substantiallysimilar nucleotide sequences encompassed by this invention are alsodefined by their ability to hybridize, under stringent conditions, withthe sequences exemplified herein. Typically, stringent conditions arethose in which the salt concentration is less than about 1.5 M Na ion(typically about 0.01 to 1.0 M Na ion concentration or other salts) atpH 7.0 to 8.3 and the temperature is at least about 30° C. for shortprobes (e.g., 10 to 50 nucleotides) and at least about 60° C. for longprobes (e.g., greater than 50 nucleotides). Stringent conditions mayalso be achieved by adding destabilizing agents such as formamide.Exemplary low stringency conditions include hybridization with a buffersolution of 6×SSC (1 M NaCl), 30 to 35% formamide, 1% SDS (sodiumdodecyl sulphate) at 37° C., and a wash in 1× to 2×SSC (20×SSC=3.0 MNaCl/0.3 M trisodium citrate) at 50 to 55° C. Exemplary moderatestringency conditions include hybridization in 6×SSC (1 M NaCl), 40 to45% formamide, 1% SDS at 37° C., and a wash in 0.5× to 1×SSC at 55 to60° C. Exemplary high stringency conditions include hybridization in6×SSC (1 M NaCl), 50% formamide, 1% SDS at 37° C., and a wash in 0.1×SSCat 60 to 65° C.

[0120] “Specificity” is typically a function of post-hybridizationwashes, the critical factors being the ionic strength and temperature ofthe final wash solution. The melting temperature T_(m) of a probe-targethybrid can be calculated to provide a starting point for determiningcorrect stringency conditions. The T_(m) is the temperature (underdefined ionic strength and pH) at which 50% of a complementary targetsequence hybridizes to a perfectly matched probe. For DNA-DNA hybrids,the T_(m) can be approximated from the equation of Meinkoth and Wahl(Anal. Biochem. (1984) 138:267-284) as follows:

[0121] T_(m)=81.5° C.+16.6 (log M)+0.41 (% G+C)-0.61 (% form)−500/L;where M is the molarity of monovalent cations, % G+C is the percentageof guanosine and cytosine nucleotides in the DNA, % form is thepercentage of formamide in the hybridization solution, and L is thelength of the hybrid in base pairs.

[0122] T_(m) is reduced by about 1° C. for each 1% of mismatching; thus,T_(m), hybridization and/or wash conditions can be adjusted to hybridizeto sequences of the desired identity. For example, if sequenceswith >90% identity are sought, the T_(m) can be decreased 110° C.Generally, stringent conditions are selected to be about 5° C. lowerthan the thermal melting point T_(m) for the specific sequence and itscomplement at a defined ionic strength and pH. However, severelystringent conditions can use a hybridization and/or wash at 1, 2, 3, or4° C. lower than the thermal melting point T_(m); moderately stringentconditions can use a hybridization and/or wash at 6, 7, 8, 9, or 10° C.lower than the thermal melting point T_(m); and low stringencyconditions can use a hybridization and/or wash at 11, 12, 13, 14, 15, or20° C. lower than the thermal melting point T_(m). Using the equation,hybridization and wash compositions, and desired T_(m), those ofordinary skill will understand that variations in the stringency ofhybridization and/or wash solutions are inherently described. If thedesired degree of mismatching results in a T_(m) of less than 45° C.(aqueous solution) or 32° C. (formamide solution) it is preferred toincrease the SSC concentration so that a higher temperature can be used.

[0123] Hybridization requires that the two nucleic acids containcomplementary sequences, although depending on the stringency of thehybridization, mismatches between bases are possible. The appropriatestringency for hybridizing nucleic acids depends on the length of thenucleic acids and the degree of complementation, variables well known inthe art. The greater the degree of similarity or homology between twonucleotide sequences, the greater the value of T_(m) for hybrids ofnucleic acids having those sequences. The relative stability(corresponding to higher T_(m)) of nucleic acid hybridizations decreasesin the following order: RNA:RNA, DNA:RNA, DNA:DNA. For hybrids ofgreater than 100 nucleotides in length, equations for calculating T_(m)have been derived (see Sambrook et al., supra, 9.50-9.51). Forhybridizations with shorter nucleic acids, i.e., oligonucleotides, theposition of mismatches becomes more important, and the length of theoligonucleotide determines its specificity (see Sambrook et al., supra,11.7-11.8). In one embodiment the length for a hybridizable nucleic acidis at least about 10 nucleotides. Preferable a minimum length for ahybridizable nucleic acid is at least about 15 nucleotides; morepreferably at least about 20 nucleotides; and most preferably the lengthis at least 30 nucleotides. Furthermore, the skilled artisan willrecognize that the temperature and wash solution salt concentration maybe adjusted as necessary according to factors such as length of theprobe and G+C composition of the target DNA.

[0124] An extensive guide to the hybridization of nucleic acids is foundin Tijssen, Laboratory Techniques in Biochemistry and MolecularBiology-Hybridization with Nucleic Acid Probes, Part I, Chapter 2“Overview of principles of hybridization and the strategy of nucleicacid probe assays” (1993) Elsevier, New York, and in Current Protocolsin Molecular Biology, (1995) Chapter 2, Ausubel et al. (Eds.), GreenePublishing and Wiley-Interscience, New York.

General Methods

[0125] All HPLC methods were performed according to Gavagan et al., J.Org Chem. (1998) 63:4792-4801.

[0126] For computational nucleic acid and protein sequence assembly andanalyses (including determination of identity and similarity),Applicants used the Wisconsin Package Version 9.0 and 10.0 GeneticsComputer Group (GCG) Gap program found in the GCG program package (usingthe Needleman and Wunsch algorithm with their standard default values ofgap creation penalty=50 and gap extension penalty=3 (Devereux et al.,Nucleic Acids Res. (1984) 12:387-395)), Sequencher Version and VectorNTI Deluxe v 4.0.3 software and database packages. Unless specifiedotherwise, all default parameters were used.

[0127] Materials and methods suitable for the maintenance and growth ofbacterial cultures are well known in the art. Techniques suitable foruse in the following examples may be found as set out in Manual ofMethods for General Bacteriology (1994) (Phillipp Gerhardt et al.(Eds.), American Society for Microbiology, Washington, D.C.) or as setout in Biotechnology: A Textbook of Industrial Microbiology (1989)Second Edition, (Thomas D. Brock, Sinauer Associates, Inc., Sunderland,Mass.).

[0128] Nitrile-containing substrates suitable for use in this inventionare dinitriles having the formula NC—R—CN where R is an alkylene grouphaving from about 1 to about 10 carbons. A more preferrednitrile-containing substrate is an aliphatic α,ω-dinitrile having theformula

NCCX_(a)(R)(CH₂)_(n)CN,

[0129] where a=0 or 1, where X=hydrogen when a=1, and R═H, alkyl orsubstituted alkyl, or alkenyl or substituted alkenyl, or alkylidene orsubstituted alkylidene, and where n=1 or 2. Most preferred for use as anitrile-containing substrate in this invention is2-methylglutaronitrile.

[0130] In an improved method for producing five-membered or six-memberedring lactams with biocatalysts comprising the biological units claimedherein, preferred is an aliphatic α,ω-dinitrile of either the formula:

[0131] where R₁ and R₂ are both H, and R₃, R₄, R₅ and R₆ are eachindependently selected from the group consisting of H, alkyl orsubstituted alkyl, or alkenyl or substituted alkenyl, or R₃ and R₄ takentogether are alkylidene or substituted alkylidene, or indepently R₅ andR₆ taken together are alkylidene or substituted alkylidene. A morepreferred nitrile-containing substrate is an aliphatic α,ω-dinitrileunsymmetrically substituted at the α-carbon atom (See U.S. Pat. No.5,858,736, incorporated herein by reference)

[0132] Procedures required for PCR amplification, DNA modifications byendo- and exo-nucleases for generating desired ends for cloning of DNA,ligations, and bacterial transformation are well known in the art.Standard recombinant DNA and molecular cloning techniques used here arewell known in the art and are described by Maniatis; and by T. J.Silhavy et al. (in Experiments with Gene Fusions, (1984) Cold SpringHarbor Laboratory Press, Cold Spring, N.Y.); and by Ausubel et al. (inCurrent Protocols in Molecular Biology (1994-1998) John Wiley & Sons,Inc., New York).

[0133] A. Isolation and Partial Amino Acid Sequencing of the Acidovoraxfacilis 72W Nitrilase Enzyme:

[0134] The nitrilase of the present invention was isolated and refinedto >90% purity from extracts of Acidovorax facilis 72W (ATCC 55746).Bacterial nitrilases are known to be generally comprised of one subunit(Novo et al., FEBS Letters (1995) 367:275-279). Methods to purifynitrilase enzymes are known in the art (Bhalla et al., Appl. Microbiol.Biotechnol. (1992) 37:184-190, Goldlust et al., Biotechnol. Appl.Biochem. (1989) 11:581-601, Yamamoto et al., Agric. Biol. Chem. (1991)55:1459-1466). The instant nitrilase was purified by passage through aQ-Sepharose ion exchange medium followed by gel filtration on a Hiload16/60 Superdex 200 column. Methods to purify and separate enzymes areknown in the art. (See, for example, Rudolph et al., Chromatogr. Sci.(1990) 51 (HPLC Biol. Macromol.):333-50.) Nitrilase activity wasmonitored during purification by measuring the rate of conversion ofbenzonitrile to benzoic acid as indicated by the increase in absorptionat 245 nm of a 5 mM solution of benzonitrile in a 100 mM, pH 7.2phosphate buffer.

[0135] N-terminal amino acid sequence and sequences of peptide digestionproducts from the purified Acidovorax facilis 72W nitrilase protein weredetermined. Nitrilase purified by gel filtration was sequenced followingtrypsin digestion using methods known to the art, including cysteinemodification by reduction followed by alkylation with 4-vinylpyridine(See, for example, Matsudaira, Methods Enzymol. (1990) 182 (GuideProtein Purif.):602-13 or Allen, Sequencing of Proteins and Peptides.In: Laboratory Techniques in Biochemistry and Molecular Biology (Burdon,R. H. and van Knippenberg, P. H., Eds), Elsevier, Amsterdam, New York,Oxford (1989)).

[0136] Database searches were run on GCG using Wisconsin SoftwarePackage 9.1 (Genetics Computer Group, Madison, Wis.). Determined aminoacid sequences were compared to protein sequence information containedin the SWISS-PROT database. Ten oligopeptides isolated from the digestof the purified Acidovorax facilis 72W nitrilase protein had amino acidsequences with >60% similarity to other known nitrilase proteins. Thisindicates that the purified protein has a high probability of containinga nitrilase enzyme.

[0137] B. Isolation of a DNA Fragment Containing the Nitrilase Gene andExpression in E. coli:

[0138] To identify possible DNA sequences with homology to knownnitrilase genes, degenerate oligonucleotide primers were designed andsynthesized for use as PCR primers. The design of these PCR primers wasbased on conserved coding regions found in bacterial nitrilase sequencesavailable from the GenBank database (GenBank Accession Numbers D12583,J03196, D13419, L32589, D67026). Genomic DNA was isolated fromAcidovorax facilis 72W (ATCC 55746) by standard methods (Maniatis)modified by adding two rounds of phenol-chloroform extraction. DNA soisolated was used as a target for PCR with numerous degenerate primercombinations. The resulting amplified products were cloned into thepGem-T vector and sequenced by methods common to the art. Fromexperiments with ten different PCR primer pairs, only one product foundin plasmid pJJ 28-5, resulting from primers 1F (SEQ ID NO:1) and 7R (SEQID NO:2), yielded a 385 bp DNA fragment (SEQ ID NO:3) with 74.1%identity to a region of the nitrilase DNA sequence from Rhodococcusrhodochrous K22 (GenBank Accession Number D12583). In addition, theputative protein product from this 385 bp DNA fragment contained anumber of peptide sequences matching those isolated from the purified72W nitrilase protein digest. This was additional evidence that theidentified 385 bp fragment contained a portion of the desired 72Wnitrilase gene.

[0139] With the discovery of this partial gene sequence with highhomology to other known nitrilase genes, Applicants have discovered apiece of DNA useful as a probe to identify recombinant clones containingthe 72W nitrilase gene in any DNA library. In addition, the successfuluse of SEQ ID NO:1 and SEQ ID NO:2 as degenerate PCR primers to discovera novel nitrilase gene shows that Applicants have discovered DNAsequences that have general utility as a means of identifying andisolating unknown nitrilases from bacterial strains.

[0140] In order to map the complete nitrilase gene to a singlerestriction fragment, Southern hybridizations were performed onAcidovorax facilis 72W genomic DNA. High molecular weight genomic DNAwas isolated from Acidovorax facilis 72W cells by using Qiagen genomictip-100/G DNA isolation kit (Qiagen Inc., USA). 1 μg genomic DNA wasdigested with a variety of restriction enzymes, resolved on 1% agarosegel followed by transfer to nylon membrane. Restriction fragmentsimmobilized on nylon membrane were probed with 385 bp partial nitrilasefragment (SEQ ID NO:3) by Southern hybridizations. The nitrilase genewas mapped to a single restriction fragment when genomic DNA wasdigested with any of the following restriction enzymes: BamHI, BclI,BglII, ClaI, EagI, KpnI, NarI, NheI, NotI, NsiI, PstI, SalI, SpeI, SstI,XbaI, XhoI. These observations provided strong evidence for the presenceof a single nitrilase gene in Acidovorax facilis 72W (ATCC 55746). EcoRIdigests, from the known partial gene sequence (SEQ ID NO:3) whichcontains an EcoRI site within itself, yielded two bands. Digests withPstI yielded a single, 4.1 kb band that reacted with the probe,suggesting that the nitrilase homolog was present on a 4.1 kb PstIfragment from the chromosome. This information was critical forselecting a restriction enzyme (in this case PstI) to generate anenriched library of genomic fragments to isolate the complete nitrilasegene.

[0141] To construct a library, Acidovorax facilis 72W genomic DNA wasdigested with PstI and resolved by gel electrophoresis. Fragmentsranging in size from 2.5-7 kilobases were recovered from the gel andligated into the vector pBluescript II SK(+) (Stratagene, La Jolla,Calif., USA). The ligated DNA was electrotransformed into E. coli DH10Belectromax cells (Life Technologies, Rockville, Md., USA). The resultantlibrary represented 4×10⁶ independent, recombinant clones of Acidovoraxfacilis 72W PstI fragments. In order to screen the library, plasmidswere prepared from cells scraped from plates.

[0142] The above plasmid library was transformed into E. coli DH5α. Alabeled probe fragment containing the 385 bp nitrilase gene fragmentfrom pJJ28-5 was used to screen the library by colony hybridization.Three colonies that showed positive hybridization with the nitrilaseprobe were grown in liquid cultures in the presence of ampicillin.Plasmids from these colonies (pnit4, pnit5, and pnit6) showed identicalrestriction fragment patterns when digested with PstI, EcoRI, andHindIII, suggesting that these plasmids contained identical inserts.PstI digestion yielded a 4.1 kb insert fragment confirming Southernhybridization results. Inserts in all the above plasmids contained oneinternal EcoRI and one HindIII site, respectively, as expected from thepartial gene sequence already identified (SEQ ID NO:3).

[0143] The inserts in plasmids pnit4, pnit5, and pnit6 had identicalnucleotide sequences (SEQ ID NO:4) containing an ORF encoding a 369amino acid sequence (SEQ ID NO:5) similar in length to other nitrilases.The primary amino acid sequence is 71% identical to the nitrilase fromRhodococcus rhodochrous K22 (GenBank Accession Number D12583). The ORFstart codon is GTG, which yields a protein beginning with a valineinstead of a more common start of methionine. The amino acid sequence(SEQ ID NO:5) encoded by the above ORF (SEQ ID NO:4) was scanned in thePROSITE database of protein families and domains using Scanprosite (www.Espay.ch/tools/scnpsitl.html) and Profilescan(www.isrec.isb-sib.ch/software/PFSCAN-form.html) and was found tocontain signature patterns conserved in all known nitrilases. Based onthis data, cysteine at position 164 (SEQ ID NOs:5 and 14) is proposed tobe the active site cysteine in Acidovorax facilis 72W nitrilase. Thesequence similarity to known nitrilases as well as the presence of thenitrilase signature together provided evidence that the gene fornitrilase was present on the 4.1 kb PstI fragment of pnit4, pnit5, andpnit6 on an ORF (SEQ ID NO:4) encoding a 369 amino acids sequence (SEQID NO:5).

[0144] The instant invention thus provides a plasmid clone DH5α:pnit4containing the complete Acidovorax facilis 72W nitrilase gene and theflanking chromosomal DNA in a form convenient for manipulation.

[0145] The Acidovorax facilis 72W nitrilase ORF identified in the 4.1 kbPstI fragment from pnit4 was cloned into a number of pet expressionvectors based on T7 promoter-T7 RNA polymerase system for overexpression(Studier et al., Meth. Enzymol. (1990) 185:60-89). More specifically,the ORF was cloned into pET-3c and pET-21a (both, Novagen, Madison,Wis.). The pET-3c nitrilase construct (pnitex2 (Example 6)) and pET-21aconstruct (pSW91(Example 7)) both have the start codon (GTG) of nativenitrilase ORF replaced with ATG. An additional pET-21a construct (pSW90(Example 7)) yields a modified form that contains an 11 amino acid T7tag fusion at the N-terminus of the protein. The plasmid pnitex2 wastransformed into two different host strains BL21 (DE3) and BL21-SIyielding strains SS1001 and SS1002 respectively (Example 6). Thesestrains allowed expression of nitrilase with or without induction byIPTG (strain SS1001, Example 6) or induction by NaCl (strain SS1002,Example 6). The plasmids pSW90 and pSW91 were transformed into BL21(DE3)yielding strains SW90 and SW91 respectively (Example 7) both of whichexpressed enzymatically active nitrilase with or without induction byIPTG. When the above E. coli strains containing these constructs wereinduced according to protocols provided by the manufacturer, SS1001,SW90, and SW91 produced a specific protein of the expected molecularweight, approximately 40 kd. In addition, when tested for nitrilaseenzyme activity (indicated by the ability to catalyze the conversion ofMGN to 4-CPA), all expression systems (SS1001, SS1002, SW90, and SW91)catalyzed this reaction, showing that the genetically engineered E. colistrains were able to produce active Acidovorax facilis 72W nitrilaseenzyme (Examples 6 and 7).

[0146] C. Production of Acidovorax facilis 72W (ATCC 55746) and E. coliExpression Strains

[0147] Growth of Acidovorax facilis Strain 72W (ATCC 55746)

[0148] One frozen seed lot vial was thawed and the 1 mL contents placedin 500 mL of sterile Inoculum Medium listed below. The inoculum wasgrown at 30° C. with shaking at 250 rpm in a 2 L flask for 24-30 h.Inoculum Medium Final Component: Concentration: Potassium phosphate,monobasic 1.5 g/L Potassium phosphate, dibasic 3.4 g/L Ammonium sulfate1.5 g/L Trisodium citrate, dihydrate 1 g/L Magnesium sulfate,heptahydrate 0.4 g/L Trace metal solution (below) 1 mL/L Amberex 695(Universal Foods) 1 g/L Glycerol (sterilized separately) 8 g/L TraceMetal Solution Stock Component: Concentration: Hydrochloric Acid 10 mL/LCalcium chloride, dihydrate 11.4 g/L Manganese Sulfate, monohydrate 1.23g/L Copper sulfate, pentahydrate 0.63 g/L Cobalt chloride, hexahydrate0.16 g/L Boric Acid 0.91 g/L Zinc sulfate, heptahydrate 1.77 g/L Sodiummolybdate, dihydrate 0.05 g/L Vanadyl sulfate, dihydrate 0.08 g/L Nickelnitrate, hexahydrate 0.04 g/L Sodium selenite 0.04 g/L Ferrous sulfate,heptahydrate 6.0 g/L

[0149] The inoculum from the shake flask was transferred aseptically toa presterilized Braun Biostat C fermenter containing the FermenterMedium listed below. Fermenter Medium Component: Final Concentration:Potassium phosphate, monobasic 0.39 g/L Potassium phosphate, dibasic0.39 g/L Difco yeast extract  5.0 g/L

[0150] Growth occurred under the following conditions: 32° C., pH6.8-7.0, dissolved oxygen at 25% of saturation. At inoculation, thefermenter contained 8.5 L of Fermenter Medium plus 218 g of NutrientFeed solution, giving a starting concentration of approximately 7 g/Lglycerol. The Nutrient Feed solution included the following componentswhich were sterilized separately and combined after cooling: potassiumphosphate, monobasic, 19.6 g in 0.25 L deionized water; magnesiumsulfate, heptahydrate, 3.3 g, plus sulfuric acid, 4 mL, in 0.15 Ldeionized water; Trace Metal solution, 67 mL, plus 400 g glycerol in0.80 L deionized water. At 18 h post inoculation, feeding of NutrientFeed solution began. Initially, the Nutrient Feed solution was added ata rate of 0.4 g feed/minute (0.15 g glycerol/min). The culture opticaldensity measured at 550 nm (O.D. 550) was approximately 8-9. At 26 h,the O.D. 550 was 16-18 and the feed rate was increased to 0.9 g feed/min(0.3 g glycerol/min). A final increase in feed rate to 1.8 g feed/min(0.6 g glycerol/min) was made at 34 h. This rate continued to the end ofthe run (about 42 h). The final O.D. 550 was approximately 65-75 andequivalent to a cell density of 25-30 g dcw/L.

[0151] Cells were recovered by centrifugation and stored frozen untiluse. For use as a biocatalyst, cells were heated to 50° C. for 1 h in0.35 M phosphate buffer (pH 7.3) and then used to catalyze thetransformation of MGN into 4-CPA.

[0152] Growth of E. coli Cells for Whole Cell Activity andImmobilization:

[0153] 25 mL of overnight culture of E. coli strain SS1001 grown from asingle colony was inoculated into 250 mL fresh LB medium. The cultureswere grown to mid-log phase and harvested by centrifugation with orwithout induction with 1 mM IPTG for 1 h. The strain SS1002 was grown inLBON (LB without NaCl) medium and induced with 0.2 M NaCl for 1 h atmid-log phase according to manufacturer's instructions for BL21-SIoverexpression strain (Life Technologies, Rockville, Md., USA). Thebacterial cell pellets were stored on ice overnight prior to measurementof nitrilase activity. For immobilization, E. coli strain SS1001 wasgrown in a 10 L batch fermentation in LB medium to an O.D. at 600 nm of8.6. Cells were harvested by centrifugation and stored on wet ice forwhole cell activities and immobilization.

[0154] Comparing the weight-specific activity of biocatalyst producedfrom the native strain, as described above, to the geneticallyengineered biocatalysts from Examples 6, 7, and 15 it is clear that theexpression strains SS1001, SS1011, and SW91 produced biocatalysts ofsubstantially greater specific activity (Table 2 in Example 6, Tables 3and 4 in Example 7, and Table 5 in Example 15) than that of the nativestrain (Tables 2, 3, 4, and 5).

EXAMPLE 1 Purification of Nitrilase Protein

[0155] All steps in this procedure were performed at 5° C. and at pH 7.5unless otherwise stated.

[0156] A 25 wt % suspension of Acidovorax facilis 72W (ATCC 55746) wetcell paste was prepared in 20 mM Tris, pH 7.5, 0.1 mM phenyl methylsulfonyl fluoride (PMSF), and 2.0 mM dithiothreitol.

[0157] An extract of this suspension was prepared by passage through aFrench press (American Instrument Co., Silver Springs, Md., USA)according to methods known to the art. Following a centrifugation at27,500 g for 30 min to remove cell debris, a 20-55% ammonium sulfatefractionation of the extract was prepared and then concentrated byovernight precipitation following the addition of solid ammonium sulfateto 65% of saturation. The concentrated protein precipitate wasreconstituted using a minimum volume of 20 mM Tris, pH 7.5 (Buffer A)and desalted over a PD10 column containing Sephadex G-25 resin(Pharmacia).

[0158] Following desalting, the concentrated protein extract wasfractionated by anion exchange chromatography using a column containing50 mL of Q-Sepharose fast flow (Pharmacia). After loading the columnwith the concentrated protein extract, the column was washed with threecolumn volumes of Buffer A at a flow rate of 2 mL/min to removeun-adsorbed protein. Adsorbed protein was eluted from the column using a0-0.5 M NaCl gradient prepared in Buffer A. Elution of protein from thecolumn was monitored at 280 nm. Nitrilase activity was monitoredthroughout purification using an assay measuring the hydrolysis ofbenzonitrile to produce benzoic acid. Nitrilase activity eluted at 0.4 MNaCl. Protein components in the 0.4 M NaCl protein fraction wereseparated by gel electrophoresis (SDS-PAGE) performed under reducingconditions (5% β-mercaptoethanol) on a 10-15% SDS polyacrylamide gel.Greater than 50% of the 0.4 M NaCl protein fraction consisted of aprotein with subunit molecular weight of 39.7 kd. This is within theexpected molecular weight range for nitrilase enzymes (Cowan et al.,Extremophiles (1998) 2:207-216). Using methods known to the art, thenative molecular weight of the nitrilase was determined to be 570 kdfollowing gel filtration chromatography in 20 mM phosphate buffer at pH7 using a Hiload 16/60 Superdex 200 column (Pharmacia) which had beencalibrated using gel filtration MW standards (Pharmacia #17-0442-01).Following gel filtration, the nitrilase protein was >90% pure. Thespecific activity of the purified enzyme was determined to be 35 IU/mgprotein using 2-methylglutaronitrile as substrate at 25° C.

EXAMPLE 2 Isolation of a DNA Fragment Showing High Homology to KnownNitrilases

[0159] Genomic DNA was isolated from Acidovorax facilis 72W (ATCC 55746)by standard methods (Maniatis) modified by adding two rounds ofphenol-chloroform extraction. DNA so isolated was used as a target foramplification by PCR using primers 1F (SEQ ID NO:1) and 7R (SEQ IDNO:2). The resulting amplified products were cloned into the pGem-Tvector (Promega, Madison, Wis.) and sequenced by methods common to theart. Clone pJJ 28-5 yielded a 385 bp DNA fragment (SEQ ID NO:3) with74.1% identity to a region of the nitrilase DNA sequence fromRhodococcus rhodochrous K 22 (GenBank Accession Number D12583).

EXAMPLE 3 Localization of a Chromosomal Fragment Containing a Sequencewith High Homology to the 385 bp Partial-Gene Fragment

[0160] 1-3 μg high molecular weight genomic DNA samples from Acidovoraxfacilis 72W were digested with restriction enzyme PstI in a buffersupplied by the manufacturer (Life Technologies (Gibco-BRL),Gaithersburg, Md., USA). Digested samples were resolved on agarose gelsand Southern-blotted on nylon membrane. Cutting at restriction sitesflanking the insert, a probe containing the 385 bp nitrilase genefragment (SEQ ID NO:3) was prepared from pJJ28-5. Hybridizations werecarried out at 60° C. followed by high stringency washes (0.1×SSC, 0.1%SDS, 60° C. for 15 min). Probe labeling, hybridization and detection fordot blots and subsequent Southern blotting experiments, were performedusing ECL random prime labeling and detection systems version II,(Amersham International plc, Buckinghamshire, England).

[0161] Digests with PstI yielded a single 4.1 kb band upon hybridizationwith the probe indicating that the nitrilase gene was present on a 4.1kb PstI fragment of the Acidovorax facilis 72W genome.

EXAMPLE 4 Construction of Acidovorax facilis 72W (ATCC 55746) GenomicLibrary

[0162] 5 μg high molecular weight genomic DNA from Acidovorax facilis72W was digested with PstI and resolved on a preparative 1% agarose gel.Fragments ranging in size from 2.5 to 7 kb were recovered from the geland ligated into PstI digested and dephosphorylated pBluescript II SK(+)vector (Stratagene, La Jolla, Calif., USA). The ligated DNA waselectrotransformed into E. coli DH10B electromax cells (LifeTechnologies (Gibco-BRL), Gaithersburg, Md., USA) and transformed cellswere plated on LB+ampicillin (100 g/mL)+IPTG+X-Gal plates. The resultantlibrary with 95% recombinant plasmids represented 4×10⁶ independentrecombinant clones. For library screening and storage, plasmids wereprepared from the cells scraped from the plates.

[0163] The above plasmid library was transformed into E. coli DH5α (LifeTechnologies, Rockville, Md., USA) cells and the transformants wereplated on LB+ampicillin 100 μg/mL plates. After incubating overnight at37° C., well-isolated colonies were transferred to nylon membrane andlysed in situ to immobilize DNA on membrane. The membranes were probedwith labeled nitrilase gene probe described in Example 3. Three coloniesthat showed positive hybridization with the nitrilase probe were grownin liquid cultures in the presence of ampicillin. Plasmids pnit4, pnit5,and pnit6 yielded identical patterns upon digestion with restrictionenzymes PstI, EcoRI, and HindIII respectively. These results confirmedthat identical inserts were present in pnit4, pnit5, and pnit6.Restriction digests yielded a 4.1 kb PstI insert as expected fromearlier Southern hybridization.

EXAMPLE 5 Sequence of Acidovorax facilis 72W (ATCC 55746) Nitrilase ORF

[0164] The 4.1 kb insert in plasmid pnit4 was sequenced using thestandard Sanger dideoxy chain termination method. Sequence analysesusing GCG MAP and Vector NTI software revealed the presence of an 1110bp open reading frame (SEQ ID NO:4; SEQ ID NO:15, bases 332-1441). ThisORF was present within a BclI-BglII fragment (SEQ ID NO:15, and FIG. 1)of the pnit4 insert. A search of GenBank sequence database revealed thatthis open reading frame (SEQ ID NO:4) had 68% identity to the nitrilaseORF from Rhodococcus rhodochrous K22 (GenBank Accession Number D12583).The 369 amino acids sequence (SEQ ID NO:5) deduced from the open readingframe (SEQ ID NO:4) is 71% identical to the Rhodococcus rhodochrous K22nitrilase protein. A scan of the amino acid sequence (SEQ ID NO:5)encoded by Acidovorax facilis 72W ORF (SEQ ID NO:4) into the PROSITEdatabase of protein families and domains using Profilescan andScanprosite tools revealed the presence of signature patterns conservedfor all known nitrilases.

EXAMPLE 6 Expression of Acidovorax facilis 72W (ATCC 55746) Nitrilase inE. coli

[0165] Oligonucleotides SEQ ID NO:6 and SEQ ID NO:7 were used in a PCRreaction to amplify the nitrilase ORF from Acidovorax facilis 72W (ATCC55746) genomic DNA. The PCR product was digested with NdeI and BamHI andwas cloned into NdeI-BamHI linearized pET-3c. The start codon ofnitrilase ORF in the resultant expression plasmid (pnitex2) is ATG (SEQID NO:13) instead of the native GTG codon (SEQ ID NO:4). Accordingly,the amino acid sequence of the nitrilase expressed from pnitex2 has M(methionine) at position 1 (SEQ ID NO:14) instead of the native V(valine) of Acidovorax facilis 72W nitrilase (SEQ ID NO:5). Plasmidspnitex2 and pET-3c (control) were transformed into E. coli strainsBL21(DE3) (Novagen, Madison, Wis., USA) to yield SS1001 and BL21(DE3):pET-3c, respectively.

[0166] Strains resulting from transforming pnitex2 and pET-3c intoBL21-SI (Life Technologies, Rockville, Md.) were named SS1002 andBL21-SI:pET-3c, respectively. Plasmid pnitex2 contains nitrilase codingsequence under the control of T7 promoter. Transcription ofchromosomally located T7 RNA polymerase, which regulates transcriptionof a gene under control of the T7 promoter (in this case the chimericnitrilase gene), is induced by the addition of IPTG in host strain BL21(DE3) and by the addition of NaCl in host BL21-SI. The above E. colitransformants, SS1001 (and the control strain BL21(DE3): pET-3c) andSS1002 (and the control strain BL21-SI: pET-3c), were grown in shakeflasks or 10 L fermenters and chimeric nitrilase gene expression wasmeasured with or without induction by adding either IPTG or NaClfollowing protocols provided by the manufacturers. Crude extracts ofstrains SS1001, SS1002, and BL21(DE3): pET-3c were prepared and run onSDS-PAGE gels with 5-20 μg total protein per lane. Molecular weight andlaser densitometer analysis of the SDS-PAGE gel determined that thestrains SS1001 and SS1002 expressed a specific protein band ofapproximately 40 kd size, corresponding to the expected size ofAcidovorax facilis 72W nitrilase protein. This band represented >50% oftotal soluble protein in SS1001. The corresponding band was absent inthe BL21(DE3): pET-3c crude extracts. Enzyme assays of the extracts fromSS1001 suggested that 12% of total soluble protein wasenzymatically-active nitrilase.

[0167] Whole cells of E. coli transformants SS1001 and SS1002, alongwith control strains BL21(DE3): pET-3c and BL21-SI: pET-3c,respectively, were also tested for nitrilase activity. The abovetransformants were grown to late log phase and the nitrilase activitylevels of whole cells with or without induction was compared to theactivity of strains harboring the control pET-3c vector only (Table 2).The nitrilase activity was measured by determining the rate of 4-CPAproduction from MGN. A 50 mg (dry cell weight)/mL cell suspension wasprepared in 0.10 M potassium phosphate buffer, pH 7.0. Into a 20-mLglass scintillation vial equipped with a magnetic stir bar was added 3.0mL of an aqueous solution of 0.40 M MGN at 25° C. With stirring, 1.0 mLof the cell suspension at 25° C. was added. At 5, 10, and 15 minutesafter the addition of the cell suspension, a 180 μL aliquot was removedfrom the reaction mixture, mixed with 5 μL of 6.0 N HCl and 20 μL of0.75 M N-methylpropionamide in water (HPLC external standard),centrifuged, and the supernatant analyzed by HPLC for the rate ofproduction of 4-CPA ammonium salt.

[0168] A unit of nitrilase activity (IU) is equivalent to production of1 micromole 4-CPA ammonium salt/min. The activity level is reported asunits per gram of dry cell weight and compared to the activity of A.facilis 72W. TABLE 2 Nitrilase Activity in E. coli transformantsNitrilase Activity Transformant Catalyst Induction (IU/g dry cellweight) E. coli SS1001 None 360 (BL21(DE3): pnitex2) E. coli SS1001 IPTG466 (BL21(DE3): pnitex2) E. coli control IPTG 0 (BL21 (DE3): pET-3c) E.coli SS1002 NaCl 288 (BL21-SI: pnitex2) E. coli control None 0 (BL21-SI:pET-3c) Acidovorax facilis 72W Not applicable 271 (ATCC 55746)

EXAMPLE 7 Expression of Acidovorax facilis 72W Nitrilase Gene in E. coli

[0169] The 72W nitrilase coding sequence was isolated from Acidovoraxfacilis 72W by PCR using primers corresponding to DNA sequencedetermined at the 5′ and 3′ ends of the coding sequence. The codingsequence was amplified using primers identified as SEQ ID NO:8 and SEQID NO:9 and subcloned into pGEM-T (Promega, Madison, Wis.). The primeridentified as SEQ ID NO:8 changes the native GTG start codon to ATG. Thenitrilase gene fragment was then removed from pGEM-T with BamHI and SacIand subcloned into the E. coli expression vector pET-21a (Novagen,Madison, Wis.), between BamHI and SacI to generate plasmid pSW90, suchthat an 11 amino acid T7 tag was encoded at the N-terminus of thenitrilase coding sequence. The coding sequence was also amplified usingprimers identified as SEQ ID NO:10 and SEQ ID NO:9 and subcloned intopGEM-T. The primer identified as SEQ ID NO:10 changes the native GTGstart codon to ATG. The gene fragment was then removed from pGEM-T withNdeI and subcloned into pET-21a at the NdeI site to generate plasmidpSW91, such that no modifications (except for the start codon asdescribed) were made to the native nitrilase coding sequence. PlasmidspSW90 and pSW91 were transformed into E. coli strain BL21(DE3) (Novagen,Madison, Wis.), to generate strains SW90 and SW91, respectively. Afterstandard growth and induction (Novagen recommendations) of SW90 andSW91, cell extracts were prepared and examined by SDS-PAGE, and a majorprotein band of the expected size, ˜40 kDa, corresponding to 72Wnitrilase protein, was observed. In the case of SW91, the nitrilaseprotein on the PAGE gel represented >50% of total soluble protein. Nocomparable major band was observed in the control.

[0170]E. coli transformant SW91 expressing the Acidovorax facilis 72W(ATCC 55746) nitrilase coding sequence was tested for nitrilase activityby measuring the rate of 4-CPA production from MGN as described inExample 6.

[0171] Following growth and IPTG induction as recommended by themanufacturer (Novagen, Madison Wis.), the nitrilase activity level ofSW91 was measured and compared to the activity of a control transformantharboring the expression vector without the nitrilase gene, and totypical activity observed for A. facilis 72W. The results are shown inTable 3. One unit of nitrilase activity (IU) is equivalent to productionof 1 micromole 4-CPA ammonium salt/min, and is reported as units pergram of dry cell weight. TABLE 3 Nitrilase activity in SW91 afterstandard growth and induction Nitrilase Activity Strain (IU/g dry cellweight) E. coli SW91 551 (BL21(DE3): pSW91) E. coli control 0 (BL21(DE3:pET-21a) Acidovorax facilis 72W 271 (ATCC 55746)

[0172]E coli transformant SW91 was also grown at 37° C. with shaking inLB media (Maniatis) without IPTG induction until saturation (12-16 h),after which cells were harvested by centrifugation and assayed fornitrilase activity. The results are shown in Table 4, and compared totypical activity observed for A. facilis 72W. TABLE 4 Nitrilase activityin SW91 after growth to saturation without induction Nitrilase ActivityStrain (IU/g dry cell weight) E. coli SW91 662 (BL21(DE3): pSW91)Acidovorax facilis 72W 271 (ATCC 55746)

EXAMPLE 8 Immobilization of Acidovorax facilis 72W or E. coliTransformant SS1001 Cells in Carrageenan

[0173] Into a 250 mL media bottle (equipped with magnetic stir bar andcontaining 64.12 g of distilled, deionized water at 50° C.) was slowlyadded 3.38 g of FMC BioPolymer ISAGEL® RG300 carrageenan with rapidstirring. The mixture was heated to 75-80° C. with rapid stirring untilthe carrageenan was completely dissolved, and the resulting solutioncooled to 55-56° C. (gelling temperature ca. 52° C.) in a thermostatedwater bath. A suspension of either Acidovorax facilis 72W cells or E.coli transformant SS1001 (12.5% dry cell weight) in 0.35 M sodiumhydrogen phosphate buffer (45 mL total volume, pH 7.3) was heated to 50°C. for 60 min (Acidovorax facilis 72W) or 12 min (E. coli transformantSS1001), then added to the carrageenan solution at 55-56° C. withstirring. The cell/carrageenan mixture was immediately added slowly to450 mL of soybean oil at 50° C. with stirring using an overhead stirrer.After cell/carrageenan droplets of the desired size were produced in theoil by controlling the stirring rate, the temperature of the oil wasreduced to 35° C. to gel the droplets, and the oil decanted from theresulting beads, which were washed with 0.10 M potassium bicarbonatebuffer (pH 7.3). A 20-gram portion of the beads were resuspended in 48.8mL of 0.10 M potassium bicarbonate buffer (pH 7.3), 0.25 g of 25 wt %glutaraldehyde in water was added, and the beads mixed for 1.0 h at 25°C. To the mixture was then added 1.0 g of 12.5 wt % polyethylenimine(BASF Lupasol® PR971L, average Mw ca. 750,000) in water and the beadsmixed for an additional hour at 25° C. The crosslinked beads were thenwashed with 50 mL of 0.30 M ammonium bicarbonate (pH 7.3) at 25° C. andstored in this same buffer at 5° C.

EXAMPLE 9 Comparison of Carrageenan-Immobilized Acidovorax facilis 72Wand E. coli Transformant SS1001 Cells as Catalyst for Production of4-Cyanopentanoic Acid Ammonium Salt

[0174] In a typical reaction, 16.5 g of immobilized cell/carrageenanbeads were placed into a 125 mL jacketed reaction vessel that wastemperature-controlled at 30° C. with a recirculating temperature bath.To the reaction vessel was added 69.25 mL of water and 14.25 mL (13.54g, 1.25 M) of 2-methyglutaronitrile, and the mixture stirred at 30° C.Samples (0.100 mL) were mixed with 0.400 mL of water, then 0.360 mL ofthe diluted sample was mixed with 0.040 mL of 0.75 MN-methylpropionamide in water and 0.020 mL of 6.0 N HCl. The resultingmixture was filtered (0.22 μm) and the filtrate analyzed by HPLC for2-methylgluratonitrile, 4-cyanopentanoic acid and 2-methlglutaric acid.The rates of production of 4-cyanopentanoic acid when usingcarrageenan-immobilized Acidovorax facilis 72W and E. coli transformantSS1001 cells were 184 mM/h and 310 mM/h, respectively. At completeconversion of 2-methylglutaronitrile, each catalyst produced4-cyanopentanoic acid ammonium salt and 2-methyl-glutaric aciddiammonium salt in 98.7% and 1.3% yields, respectively.

EXAMPLE 10 Immobilization of E. coli Transformant SW91 Cells in CalciumAlginate

[0175] Into a 100 mL media bottle (equipped with magnetic stir bar andcontaining 22.9 g of distilled, deionized water at 50° C.) was slowlyadded 1.10 g of FMC BioPolymer Protanal® LF 10/60 alginate with rapidstirring. The mixture was heated to 75-80° C. with rapid stirring untilthe alginate was completely dissolved, and the resulting solution cooledto 25° C. in a water bath. A suspension of E. coli transformant SW91(50% wet cell weight, 11.5% dry cell weight) in 0.15 M sodium acetatebuffer (16 mL total volume, pH 7.0) was added to the alginate solutionat 25° C. with stirring. The cell/alginate mixture was added dropwise bysyringe to 213 mL of 0.20 M calcium acetate buffer (pH 7.0) at 25° C.with stirring. After stirring for 2 h, the buffer was decanted from theresulting beads, which were resuspended in 84 mL of 0.20 M calciumacetate buffer (pH 7.0) at 25° C. With stirring, 0.88 g of 25 wt %glutaraldehyde in water was added and the beads mixed for 1.0 h at 25°C. To the mixture was then added 3.5 g of 12.5 wt % polyethylenimine(BASF Lupasol® PR971L, average Mw ca. 750,000) in water and the beadsmixed for an additional 1 h at 25° C. The crosslinked beads were thenwashed twice with 84 mL of 0.20 M calcium acetate buffer (pH 7.0) at 25°C. and stored in this same buffer at 5° C.

EXAMPLE 11 Calcium Alginate-Immobilized E. coli Transformant SW91 Cellsas Catalyst for Production of 4-Cyanopentanoic Acid Ammonium Salt

[0176] Into a 125 mL jacketed reaction vessel (temperature-controlled at30° C. with a recirculating temperature bath) was placed 16.5 g of E.coli SW91/alginate beads prepared as described in Example 10. To thereaction vessel was added 68.25 mL of water, 1.0 mL of 0.20 M calciumacetate buffer (pH 7.0), and 14.25 mL (13.54 g, 1.25 M) of2-methyglutaronitrile, and the mixture stirred at 30° C. Samples (0.100mL) were mixed with 0.400 mL of water, then 0.360 mL of the dilutedsample was mixed with 0.040 mL of 0.75 M N-methylpropionamide in waterand 0.020 mL of 6.0 N HCl. The resulting mixture was filtered (0.22 μm)and the filtrate analyzed by HPLC for 2-methylgluratonitrile,4-cyanopentanoic acid and 2-methlglutaric acid. The rate of productionof 4-cyanopentanoic acid was 739 mM/h, and the reaction was complete inless than 2.5 h. At complete conversion of 2-methylglutaronitrile, theyields of 4-cyanopentanoic acid ammonium salt and 2-methylglutaric aciddiammonium salt were 98.5% and 1.5% yields, respectively.

[0177] At the end of the reaction the product mixture was decanted fromthe catalyst beads, which were reused in an additional five consecutivebatch reactions as described above. The rate of production of4-cyanopentanoic acid in reaction six was 721 mM/h, and the reaction wascomplete in less than 2.5 h. At complete conversion of2-methylglutaronitrile, the yields of 4-cyanopentanoic acid ammoniumsalt and 2-methylglutaric acid diammonium salt were 98.0% and 2.0%yields, respectively.

EXAMPLE 12 Total Turnover Number (TTN) for Immobilized Acidovoraxfacilis 72W Cell Nitrilase

[0178] Into a 125 mL jacketed reaction vessel (temperature-controlled at25° C.) was placed 16.5 g of immobilized Acidovorax facilis cellcatalyst (prepared as described in Example 8). To the reaction vesselwas added 72.1 mL of water and 11.4 mL (10.83 g, 1.0 M) of MGN, and themixture stirred at 25° C. Samples (0.100 mL) were mixed with 0.400 mL ofwater, then 0.360 mL of a diluted sample was mixed with 0.040 mL of 0.75M N-methylpropionamide in water and 0.020 mL of 6.0 N HCl. The resultingmixture was filtered (0.22 μm) and the filtrate analyzed by HPLC. Aftercomplete conversion of MGN, the product mixture and catalyst weredecanted to a tared 250 mL beaker and the aqueous product mixturedecanted from the catalyst. The remaining catalyst and product mixtureheel was weighed and the weight recorded, then water was added to thecatalyst to a final total weight (water and catalyst) of 88.6 g. Thecatalyst suspension was transferred back to the reaction vessel, 11.4 mLof MGN added, and the reaction repeated.

[0179] A total of sixty-seven consecutive batch reactions with catalystrecycle were run. There was no measurable loss of catalyst bead weightover the course of the sixty-seven recycle reactions, and 1001 g 4-CPA/gdcw Acidovorax facilis 72W cells were produced. The initial reactionrate was 142 mM 4-CPA/h, which corresponds to 237 IU of nitrilaseactivity, or 6.76 mg (1.7×10⁻⁷ mole) of 72W nitrilase. The amount of4-CPA produced in 67 consecutive batch reactions was 6.6 moles, thus thetotal turnover number (TTN=moles product per moles enzyme) was6.6/1.7×10⁻⁷, or 3.9×10⁷ TTN.

EXAMPLE 13 Expression of Acidovorax facilis 72W Nitrilase CodingSequence in Pichia pastoris

[0180] A synthetic nitrilase gene (SEQ ID NO:16) (which encodes aprotein sequence identical to that found in Acidovorax facilis 72W (SEQID NO:14)) was constructed using sixteen 90 bp oligomers (SEQ IDNOS:17-32) and the technique of PCR-mediated overlap extension. Thesynthetic gene optimizes the codon usage found in Pichia pastoris, whichis considerably different from that found in the A. facilis 72Wnitrilase gene sequence. After confirming by nucleotide sequencing, thesynthetic nitrilase gene is subcloned into pGAPZA and into pPICZA(Invitrogen, San Diego, Calif.) at the EcoRI site. pGAPZA uses theconstitutive P. pastoris GAP (glyceraldehyde-3-phosphate dehydrogenase)promoter to drive expression of foreign genes; pPICZA uses the methanolinducible P. pastoris AOXI (alcohol oxidase I) promoter to driveexpression of foreign genes. Plasmids pGAP::nit and pAOX::nit are usedto transform P. pastoris GS115 (Invitrogen) to zeocin resistance byspheroplast transformation essentially as described (Cregg et al., Mol.Cell Biol. (1985) 5:3376-3385). After appropriate growth and induction(only pAOX::nit requires induction), transformants producing nitrilaseare identified by SDS-PAGE protein analysis of cells extracts preparedessentially as described (Sreekrishna et al., Biochem (1989)28:4117-4125), and by enzyme activity assay as previously described(Examples 6 and 7).

EXAMPLE 14 Determination of Acidovorax facilis 72W Nitrilase as % ofTotal Soluble Protein

[0181] A crude extract of Acidovorax facilis 72W was prepared by passageof a 25 wt % cell suspension through a french press as previouslydescribed (Example 1). Protein components in an aliquot of the solubleprotein fraction of the crude extract were separated by gelelectrophoresis (SDS-PAGE) performed under reducing conditions (5%P-mercaptoethanol) on a 10-15% SDS polyacrylamide gel. Followingelectrophoresis, the gel was treated for 15 min at 25° C. with asolution composed of 0.2% Coomassie Brilliant Blue dye, 30% methanol,and 10% acetic acid. Protein bands were visualized in the gel followingde-staining with a solution composed of 30% methanol and 10% aceticacid. The protein bands in the gel were then integrated using an LKBUltroscan XL Enhanced Laser Densitometer. The nitrilase band having asubunit MW of ca. 40 kd represented 3.4% of the total soluble proteindetected on the gel.

EXAMPLE 15 Construction of SS1011 [MG1655(DE3):pnitex2] and itsNitrilase Activity

[0182] λDE3 prophage was site-specifically integrated into thechromosome of E. coli strain MG1655 (ATCC 47076) to yield strainMG1655(DE3). λDE3 lysogenization kit (Catalog No 69734-3, Novagen, Inc.Madison, Wis.) was used for this purpose according to manufacturer'sinstructions. MG1655(DE3) was transformed with plasmid pnitex2 describedin Example 6 to yield SS1011. Strains SS1011 and SS1001 were grown in LBmedium for 16-17 h. Whole cell nitrilase activity as shown in Table 5was determined using a microliter plate-based spectrophometric assayperformed at 35° C. that measures the hydrolysis of benzonitrile (9.5mM, 0.1 M phosphate buffer, pH 7.0) to produce benzoic acid as indicatedby the increase in absorption at 245 nm. The nitrilase activity units(IU) determined using this assay are typically 5-6 fold higher thanthose determined using the previously described methylglutaronitrileassay due to a combination of higher assay temperature (35° C. vs. 25°C.) and the relatively higher substrate specificity of the enzyme forbenzonitrile. TABLE 5 Nitrilase activity in E. coli transformants SS1001and SS1011 determined by assay of hydrolysis of benzonitrile NitrilaseActivity Transformant Catalyst (IU/g dry cell weight) E. coli SS10113652 (MG1655(DE3): pnitex2) E. coli SS1001 4500 (BL21(DE3): pnitex2)Acidovorax facilis 72W 1491 (ATCC 55746)

[0183]

1 32 1 17 DNA Artificial Sequence Description of Artificial SequenceForward primer (1F) 1 tkkmtkccsg gctaycc 17 2 17 DNA Artificial SequenceDescription of Artificial Sequence Reverse primer (7R) 2 ggccasshtgmrayrtg 17 3 385 DNA Acidovorax facilis 3 ctattgggcg tggctcggcgacgtgaagta cagcctaagc tttacttcac gctatcacga 60 gaattcgttg gagctaggtgacgaccgtat gcgtcgcctc cagctggccg cgcgccgcaa 120 caaaatcgca ctcgtcatgggctattcgga gcgggaagcc ggatcgcgct atctgagcca 180 ggtgttcatc gacgagcgtggcgagatcgt tgccaatcgg cgcaagctga agcccacaca 240 cgttgagcgt acgatctacggcgaaggcaa cggaaccgat ttcctcacgc acgacttcgc 300 gttcggacgc gtcggtggattgaactgctg ggaacatttc caaccgctca gcaagttcat 360 gatgtacagc ctcggtgagcaggtc 385 4 1110 DNA Acidovorax facilis 4 gtggtttcgt ataacagcaagttcctcgcg gcaaccgttc aggcagagcc ggtatggctc 60 gacgcagacg caacgatcgacaagtcgatc ggcatcatcg aagaagctgc ccaaaagggc 120 gcgagtctga tcgctttcccggaagtattc attccgggct acccctattg ggcgtggctc 180 ggcgacgtga agtacagcctaagctttact tcacgctatc acgagaattc gttggagcta 240 ggtgacgacc gtatgcgtcgcctccagctg gccgcgcgcc gcaacaaaat cgcactcgtc 300 atgggctatt cggagcgggaagccggatcg cgctatctga gccaggtgtt catcgacgag 360 cgtggcgaga tcgttgccaatcggcgcaag ctgaagccca cacacgttga gcgtacgatc 420 tacggcgaag gcaacggaaccgatttcctc acgcacgact tcgcgttcgg acgcgtcggt 480 ggattgaact gctgggaacatttccaaccg ctcagcaagt tcatgatgta cagcctcggt 540 gagcaggtcc acgttgcatcgtggccggcg atgtcccctc ttcagccgga tgttttccaa 600 ctgagcatcg aagccaacgcgacggtcacc cgctcgtacg caatcgaagg ccaaaccttt 660 gtgctttgct cgacgcaggtgatcggacct agcgcgatcg aaacgttctg cctcaacgac 720 gaacagcgcg cactgttgccgcaaggatgt ggctgggcgc gcatttacgg cccggatgga 780 agcgagcttg cgaagcctctggcggaagat gctgagggga tcttgtacgc agagatcgat 840 ctggagcaga ttctgctggcgaaggctgga gccgatccgg tcgggcacta ttcgcggcct 900 gacgtgctgt cggtccagttcgacccgcgc aatcatacgc cagttcatcg catcggcatt 960 gacggtcgct tggatgtgaatacccgcagt cgcgtggaga atttccgact gcgacaagcg 1020 gctgagcagg agcgtcaggcatccaagcgg ctcggaacga aactctttga acaatccctt 1080 ctggctgaag aaccggtcccagcaaagtag 1110 5 369 PRT Acidovorax facilis 5 Val Val Ser Tyr Asn SerLys Phe Leu Ala Ala Thr Val Gln Ala Glu 1 5 10 15 Pro Val Trp Leu AspAla Asp Ala Thr Ile Asp Lys Ser Ile Gly Ile 20 25 30 Ile Glu Glu Ala AlaGln Lys Gly Ala Ser Leu Ile Ala Phe Pro Glu 35 40 45 Val Phe Ile Pro GlyTyr Pro Tyr Trp Ala Trp Leu Gly Asp Val Lys 50 55 60 Tyr Ser Leu Ser PheThr Ser Arg Tyr His Glu Asn Ser Leu Glu Leu 65 70 75 80 Gly Asp Asp ArgMet Arg Arg Leu Gln Leu Ala Ala Arg Arg Asn Lys 85 90 95 Ile Ala Leu ValMet Gly Tyr Ser Glu Arg Glu Ala Gly Ser Arg Tyr 100 105 110 Leu Ser GlnVal Phe Ile Asp Glu Arg Gly Glu Ile Val Ala Asn Arg 115 120 125 Arg LysLeu Lys Pro Thr His Val Glu Arg Thr Ile Tyr Gly Glu Gly 130 135 140 AsnGly Thr Asp Phe Leu Thr His Asp Phe Ala Phe Gly Arg Val Gly 145 150 155160 Gly Leu Asn Cys Trp Glu His Phe Gln Pro Leu Ser Lys Phe Met Met 165170 175 Tyr Ser Leu Gly Glu Gln Val His Val Ala Ser Trp Pro Ala Met Ser180 185 190 Pro Leu Gln Pro Asp Val Phe Gln Leu Ser Ile Glu Ala Asn AlaThr 195 200 205 Val Thr Arg Ser Tyr Ala Ile Glu Gly Gln Thr Phe Val LeuCys Ser 210 215 220 Thr Gln Val Ile Gly Pro Ser Ala Ile Glu Thr Phe CysLeu Asn Asp 225 230 235 240 Glu Gln Arg Ala Leu Leu Pro Gln Gly Cys GlyTrp Ala Arg Ile Tyr 245 250 255 Gly Pro Asp Gly Ser Glu Leu Ala Lys ProLeu Ala Glu Asp Ala Glu 260 265 270 Gly Ile Leu Tyr Ala Glu Ile Asp LeuGlu Gln Ile Leu Leu Ala Lys 275 280 285 Ala Gly Ala Asp Pro Val Gly HisTyr Ser Arg Pro Asp Val Leu Ser 290 295 300 Val Gln Phe Asp Pro Arg AsnHis Thr Pro Val His Arg Ile Gly Ile 305 310 315 320 Asp Gly Arg Leu AspVal Asn Thr Arg Ser Arg Val Glu Asn Phe Arg 325 330 335 Leu Arg Gln AlaAla Glu Gln Glu Arg Gln Ala Ser Lys Arg Leu Gly 340 345 350 Thr Lys LeuPhe Glu Gln Ser Leu Leu Ala Glu Glu Pro Val Pro Ala 355 360 365 Lys 6 27DNA Artificial Sequence Description of Artificial SequencePrimer 6gacgcatatg gtttcgtata acagcaa 27 7 28 DNA Artificial SequenceDescription of Artificial Sequence Primer 7 cgacggatcc ttatggctactttgctgg 28 8 31 DNA Artificial Sequence Description of ArtificialSequencePrimer 8 cggatccatg gtttcgtata acagcaagtt c 31 9 23 DNAArtificial Sequence Description of Artificial SequencePrimer 9ttatggctac tttgctggga ccg 23 10 29 DNA Artificial Sequence Descriptionof Artificial Sequence Primer 10 tacatatggt ttcgtataac agcaagttc 29 1126 DNA Artificial Sequence Description of Artificial Sequence Primer 11catctcgaga tggtttcgta taacag 26 12 23 DNA Artificial SequenceDescription of Artificial Sequence Primer 12 cactcgagct actttgctgg gac23 13 1110 DNA Acidovorax facilis 13 atggtttcgt ataacagcaa gttcctcgcggcaaccgttc aggcagagcc ggtatggctc 60 gacgcagacg caacgatcga caagtcgatcggcatcatcg aagaagctgc ccaaaagggc 120 gcgagtctga tcgctttccc ggaagtattcattccgggct acccctattg ggcgtggctc 180 ggcgacgtga agtacagcct aagctttacttcacgctatc acgagaattc gttggagcta 240 ggtgacgacc gtatgcgtcg cctccagctggccgcgcgcc gcaacaaaat cgcactcgtc 300 atgggctatt cggagcggga agccggatcgcgctatctga gccaggtgtt catcgacgag 360 cgtggcgaga tcgttgccaa tcggcgcaagctgaagccca cacacgttga gcgtacgatc 420 tacggcgaag gcaacggaac cgatttcctcacgcacgact tcgcgttcgg acgcgtcggt 480 ggattgaact gctgggaaca tttccaaccgctcagcaagt tcatgatgta cagcctcggt 540 gagcaggtcc acgttgcatc gtggccggcgatgtcccctc ttcagccgga tgttttccaa 600 ctgagcatcg aagccaacgc gacggtcacccgctcgtacg caatcgaagg ccaaaccttt 660 gtgctttgct cgacgcaggt gatcggacctagcgcgatcg aaacgttctg cctcaacgac 720 gaacagcgcg cactgttgcc gcaaggatgtggctgggcgc gcatttacgg cccggatgga 780 agcgagcttg cgaagcctct ggcggaagatgctgagggga tcttgtacgc agagatcgat 840 ctggagcaga ttctgctggc gaaggctggagccgatccgg tcgggcacta ttcgcggcct 900 gacgtgctgt cggtccagtt cgacccgcgcaatcatacgc cagttcatcg catcggcatt 960 gacggtcgct tggatgtgaa tacccgcagtcgcgtggaga atttccgact gcgacaagcg 1020 gctgagcagg agcgtcaggc atccaagcggctcggaacga aactctttga acaatccctt 1080 ctggctgaag aaccggtccc agcaaagtag1110 14 369 PRT Acidovorax facilis 14 Met Val Ser Tyr Asn Ser Lys PheLeu Ala Ala Thr Val Gln Ala Glu 1 5 10 15 Pro Val Trp Leu Asp Ala AspAla Thr Ile Asp Lys Ser Ile Gly Ile 20 25 30 Ile Glu Glu Ala Ala Gln LysGly Ala Ser Leu Ile Ala Phe Pro Glu 35 40 45 Val Phe Ile Pro Gly Tyr ProTyr Trp Ala Trp Leu Gly Asp Val Lys 50 55 60 Tyr Ser Leu Ser Phe Thr SerArg Tyr His Glu Asn Ser Leu Glu Leu 65 70 75 80 Gly Asp Asp Arg Met ArgArg Leu Gln Leu Ala Ala Arg Arg Asn Lys 85 90 95 Ile Ala Leu Val Met GlyTyr Ser Glu Arg Glu Ala Gly Ser Arg Tyr 100 105 110 Leu Ser Gln Val PheIle Asp Glu Arg Gly Glu Ile Val Ala Asn Arg 115 120 125 Arg Lys Leu LysPro Thr His Val Glu Arg Thr Ile Tyr Gly Glu Gly 130 135 140 Asn Gly ThrAsp Phe Leu Thr His Asp Phe Ala Phe Gly Arg Val Gly 145 150 155 160 GlyLeu Asn Cys Trp Glu His Phe Gln Pro Leu Ser Lys Phe Met Met 165 170 175Tyr Ser Leu Gly Glu Gln Val His Val Ala Ser Trp Pro Ala Met Ser 180 185190 Pro Leu Gln Pro Asp Val Phe Gln Leu Ser Ile Glu Ala Asn Ala Thr 195200 205 Val Thr Arg Ser Tyr Ala Ile Glu Gly Gln Thr Phe Val Leu Cys Ser210 215 220 Thr Gln Val Ile Gly Pro Ser Ala Ile Glu Thr Phe Cys Leu AsnAsp 225 230 235 240 Glu Gln Arg Ala Leu Leu Pro Gln Gly Cys Gly Trp AlaArg Ile Tyr 245 250 255 Gly Pro Asp Gly Ser Glu Leu Ala Lys Pro Leu AlaGlu Asp Ala Glu 260 265 270 Gly Ile Leu Tyr Ala Glu Ile Asp Leu Glu GlnIle Leu Leu Ala Lys 275 280 285 Ala Gly Ala Asp Pro Val Gly His Tyr SerArg Pro Asp Val Leu Ser 290 295 300 Val Gln Phe Asp Pro Arg Asn His ThrPro Val His Arg Ile Gly Ile 305 310 315 320 Asp Gly Arg Leu Asp Val AsnThr Arg Ser Arg Val Glu Asn Phe Arg 325 330 335 Leu Arg Gln Ala Ala GluGln Glu Arg Gln Ala Ser Lys Arg Leu Gly 340 345 350 Thr Lys Leu Phe GluGln Ser Leu Leu Ala Glu Glu Pro Val Pro Ala 355 360 365 Lys 15 1776 DNAAcidovorax delafieldii 15 tgatcactcc tgaccacctt gctgaaaaat tcagcagcgtagccgtcaac gtggctcaat 60 tttcagtgtg atccacccca aaaatggccc agttttcgttgtgattcaac agcgtcgtgt 120 ctatgacgtc tactccatac tttcgcaaga aaaagggggggaaatttttc attccccaat 180 tattagggag atcggtctaa tagtaaaggg caaaccctgattttttatta ggctagatgg 240 tctaataatt aaatcagctc ggcgaatgcg tagcgctcgggcaacccaga caaggcaatt 300 ctgacagtga cacccctctt aggagacgac cgtggtttcgtataacagca agttcctcgc 360 ggcaaccgtt caggcagagc cggtatggct cgacgcagacgcaacgatcg acaagtcgat 420 cggcatcatc gaagaagctg cccaaaaggg cgcgagtctgatcgctttcc cggaagtatt 480 cattccgggc tacccctatt gggcgtggct cggcgacgtgaagtacagcc taagctttac 540 ttcacgctat cacgagaatt cgttggagct aggtgacgaccgtatgcgtc gcctccagct 600 ggccgcgcgc cgcaacaaaa tcgcactcgt catgggctattcggagcggg aagccggatc 660 gcgctatctg agccaggtgt tcatcgacga gcgtggcgagatcgttgcca atcggcgcaa 720 gctgaagccc acacacgttg agcgtacgat ctacggcgaaggcaacggaa ccgatttcct 780 cacgcacgac ttcgcgttcg gacgcgtcgg tggattgaactgctgggaac atttccaacc 840 gctcagcaag ttcatgatgt acagcctcgg tgagcaggtccacgttgcat cgtggccggc 900 gatgtcccct cttcagccgg atgttttcca actgagcatcgaagccaacg cgacggtcac 960 ccgctcgtac gcaatcgaag gccaaacctt tgtgctttgctcgacgcagg tgatcggacc 1020 tagcgcgatc gaaacgttct gcctcaacga cgaacagcgcgcactgttgc cgcaaggatg 1080 tggctgggcg cgcatttacg gcccggatgg aagcgagcttgcgaagcctc tggcggaaga 1140 tgctgagggg atcttgtacg cagagatcga tctggagcagattctgctgg cgaaggctgg 1200 agccgatccg gtcgggcact attcgcggcc tgacgtgctgtcggtccagt tcgacccgcg 1260 caatcatacg ccagttcatc gcatcggcat tgacggtcgcttggatgtga atacccgcag 1320 tcgcgtggag aatttccgac tgcgacaagc ggctgagcaggagcgtcagg catccaagcg 1380 gctcggaacg aaactctttg aacaatccct tctggctgaagaaccggtcc cagcaaagta 1440 gccataagtt gagagtcgcg agatagtatc ggggaaagccatctctggtc ttccccttta 1500 ttctccaagc cgacatcacc gctgaaagcg ggtttctttgctaccccgag tttcgatccc 1560 gcatcgccgt cgcgtgagat ttgcgtcaga gcggacattcaagttgtgtg gcaaggtcgt 1620 ccagactgtc cacggaaaat tcccagttct cactcggttcaaggtcagtc gtttgctgcg 1680 ggccgtgttc ctgtggccgc ctgacgaatg ccgtcctcaggccacaacgt cgagcggctg 1740 ccaagtcatc gttgtgcgcc gccaccatgc agatct 177616 1110 DNA Artificial Sequence Description of Artificial Sequence Asynthetic version of the nitrilase gene 16 atggtttctt acaactccaagttcttggct gctactgttc aagctgagcc agtttggttg 60 gacgcagacg ctactattgacaagtctatc ggtatcatcg aagaagctgc ccaaaagggt 120 gcctctttga tcgctttcccagaagttttc attccaggtt acccatactg ggcctggttg 180 ggtgacgtta agtactctttgtcctttact tccagatatc acgagaactc tttggagttg 240 ggtgacgaca gaatgcgtagactgcaattg gctgcccgta gaaacaaaat tgctttggtc 300 atgggttatt ccgagagagaagctggatct cgttacttgt cccaagtctt catcgacgag 360 agaggtgaga ttgttgcaaatcgtcgtaag ttgaagccaa ctcacgttga gcgtaccatc 420 tacggagaag gtaacggaaccgatttcttg actcacgact tcgccttcgg aagagttggt 480 ggattgaact gttgggaacatttccaacct ctgtctaagt tcatgatgta ctccttgggt 540 gagcaagtcc acgttgcttcttggccagct atgtcccctc ttcagccaga tgttttccaa 600 ttgtccatcg aagccaacgccaccgtcacc agatcctacg ccatcgaagg tcaaactttt 660 gtcctttgct ctacccaggtcattggacct tctgctatcg aaaccttctg tctgaacgac 720 gaacagagag ctttgttgccacaaggatgt ggttgggcaa gaatttacgg tccagatgga 780 tctgagcttg ccaagcctttggctgaagat gctgagggta ttttgtacgc tgagatcgat 840 ttggagcaaa ttctgctggccaaggctgga gccgatccag tcggtcacta ctccagacct 900 gacgtcttgt ccgtccagttcgaccctaga aaccacactc cagttcacag aattggtatt 960 gacggtagat tggatgttaacaccagatcc agagtcgaga acttcagact gagacaagct 1020 gctgagcagg agagacaggcttctaagaga cttggaacta aacttttcga acaatctctt 1080 ttggctgaag aacctgtcccagccaagtaa 1110 17 84 DNA Artificial Sequence Description of ArtificialSequenceSynthetic oligonucleotide 17 catgaattca tggtttctta caactccaagttcttggctg ctactgttca agctgagcca 60 gtttggttgg acgcagacgc tact 84 18 90DNA Artificial Sequence Description of Artificial SequenceSyntheticoligonucleotide 18 tttgatcgct ttcccagaag ttttcattcc aggttacccatactgggcct ggttgggtga 60 cgttaagtac tctttgtcct ttacttccag 90 19 90 DNAArtificial Sequence Description of Artificial SequenceSyntheticoligonucleotide 19 aattggctgc ccgtagaaac aaaattgctt tggtcatgggttattccgag agagaagctg 60 gatctcgtta cttgtcccaa gtcttcatcg 90 20 90 DNAArtificial Sequence Description of Artificial SequenceSyntheticOligonucleotide 20 gttgagcgta ccatctacgg agaaggtaac ggaaccgatttcttgactca cgacttcgcc 60 ttcggaagag ttggtggatt gaactgttgg 90 21 90 DNAArtificial Sequence Description of Artificial Sequence Syntheticoligonucleotide 21 agtccacgtt gcttcttggc cagctatgtc ccctcttcagccagatgttt tccaattgtc 60 catcgaagcc aacgccaccg tcaccagatc 90 22 90 DNAArtificial Sequence Description of Artificial Sequence Syntheticoligonucleotide 22 gaccttctgc tatcgaaacc ttctgtctga acgacgaacagagagctttg ttgccacaag 60 gatgtggttg ggcaagaatt tacggtccag 90 23 90 DNAArtificial Sequence Description of Artificial Sequence Syntheticoligonucleotide 23 tacgctgaga tcgatttgga gcaaattctg ctggccaaggctggagccga tccagtcggt 60 cactactcca gacctgacgt cttgtccgtc 90 24 90 DNAArtificial Sequence Description of Artificial Sequence Syntheticoligonucleotide 24 tagattggat gttaacacca gatccagagt cgagaacttcagactgagac aagctgctga 60 gcaggagaga caggcttcta agagacttgg 90 25 84 DNAArtificial Sequence Description of Artificial SequenceSyntheticoligonucleotide 25 catgaattct tacttggctg ggacaggttc ttcagccaaaagagattgtt cgaaaagttt 60 agttccaagt ctcttagaag cctg 84 26 90 DNAArtificial Sequence Description of Artificial SequenceSyntheticoligonucleotide 26 tggtgttaac atccaatcta ccgtcaatac caattctgtgaactggagtg tggtttctag 60 ggtcgaactg gacggacaag acgtcaggtc 90 27 90 DNAArtificial Sequence Description of Artificial SequenceSyntheticoligonucleotide 27 tccaaatcga tctcagcgta caaaataccc tcagcatcttcagccaaagg cttggcaagc 60 tcagatccat ctggaccgta aattcttgcc 90 28 90 DNAArtificial Sequence Description of Artificial SequenceSyntheticoligonucleotide 28 ggtttcgata gcagaaggtc caatgacctg ggtagagcaaaggacaaaag tttgaccttc 60 gatggcgtag gatctggtga cggtggcgtt 90 29 90 DNAArtificial Sequence Description of Artificial SequenceSyntheticoligonucleotide 29 gccaagaagc aacgtggact tgctcaccca aggagtacatcatgaactta gacagaggtt 60 ggaaatgttc ccaacagttc aatccaccaa 90 30 90 DNAArtificial Sequence Description of Artificial Sequence Syntheticoligonucleotide 30 ccgtagatgg tacgctcaac gtgagttggc ttcaacttacgacgatttgc aacaatctca 60 cctctctcgt cgatgaagac ttgggacaag 90 31 90 DNAArtificial Sequence Description of Artificial Sequence Syntheticoligonucleotide 31 gtttctacgg gcagccaatt gcagtctacg cattctgtcgtcacccaact ccaaagagtt 60 ctcgtgatat ctggaagtaa aggacaaaga 90 32 90 DNAArtificial Sequence Description of Artificial SequenceSyntheticoligonucleotide 32 cttctgggaa agcgatcaaa gaggcaccct tttgggcagcttcttcgatg ataccgatag 60 acttgtcaat agtagcgtct gcgtccaacc 90

What is claimed is:
 1. An isolated nucleic acid fragment encoding anitrilase enzyme selected from the group consisting of: (a) an isolatednucleic acid fragment encoding all or a substantial portion of the aminoacid sequence selected from the group consisting of SEQ ID NO:5 and SEQID NO:14; (b) an isolated nucleic acid fragment that is substantiallysimilar to an isolated nucleic acid fragment encoding all or asubstantial portion of the amino acid sequence selected from the groupconsisting of SEQ ID NO:5 and SEQ ID NO:14; (c) an isolated nucleic acidmolecule that hybridizes with the isolated nucleic acid fragment of (a)under hybridization conditions of 6×SSC (1M NaCl), 40 to 45% formamide,1% SDS at 37° C., and a wash in 0.5× to 1×SSC at 55 to 60° C.; and (d)an isolated nucleic acid fragment that is completely complementary to(a), (b) or (c).
 2. An isolated nucleic acid fragment comprising a firstnucleotide sequence encoding a polypeptide of at least 369 amino acidsthat has greater than 71% identity based on the Needleman and Wunschalgorithm when compared to a polypeptide encoded by the sequenceidentified in SEQ ID NO:5, or a second nucleotide sequence comprisingthe complement of the first nucleotide sequence.
 3. An isolated nucleicacid fragment encoding a nitrilase enzyme, or a fragment thereof,selected from the group consisting of: (a) an isolated nucleic acidfragment selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2,SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ IDNO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ IDNO:15 and SEQ ID NO:16; (b) an isolated nucleic acid molecule thathybridizes with the isolated nucleic acid fragment of (a) underhybridization conditions of 6×SSC (1M NaCl), 40 to 45% formamide, 1% SDSat 37° C., and a wash in 0.5× to 1×SSC at 55 to 60° C.; and (c) anisolated nucleic acid fragment that is completely complementary to (a)or (b).
 4. An isolated nucleic acid sequence encoding a nitrilase enzymeselected from the group consisting of SEQ ID NO:4, SEQ ID NO:13, SEQ IDNO:15, and SEQ ID NO:16.
 5. The isolated nucleic acid fragment of any ofclaims 1, 2, 3, or 4 wherein the fragment is isolated from an Acidovoraxstrain.
 6. A polypeptide encoded by the nucleic acid fragments of any ofclaims 1, 2, 3, or
 4. 7. A polypeptide according to claim 6 having theamino acid sequence selected from the group consisting of SEQ ID NO:5and SEQ ID NO:14.
 8. The polypeptide of claim 6 further characterized bynitrilase activity on nitrile-containing substrates selected from thegroup consisting of aliphatic nitriles and aromatic nitriles.
 9. Achimeric gene comprising the isolated nucleic acid fragment of any ofclaims 1, 2, 3, 4 or 5 operably linked to suitable regulatory sequences.10. A plasmid pSW91 contained in E. coli SW91 having the designationATCC PTA-1175, a plasmid pnit4 contained in E. coli DH5 α: pnit4 havingthe designation ATCC PTA-1176, or a plasmid pnitex2 contained in eitherE. coli SS1002 or in E. coli SS1011.
 11. An expression cassettecomprising the chimeric gene of claim
 9. 12. The expression cassette ofclaim 11 selected from the group consisting of the plasmids pSW91,pnit4, and pnitex2.
 13. A transformed microorganism comprising thechimeric gene claim
 9. 14. A transformed microorganism comprising theplasmid of claim
 10. 15. A transformed microorganism comprising theexpression cassette of claim
 11. 16. The transformed microorganism ofclaim 15 wherein the expression cassette is chromosomally integrated.17. The transformed microorganism of claim 16 further comprisingsuitable regulatory sequences.
 18. The transformed microorganism ofclaim 17 wherein the suitable regulatory sequences comprise a) at leastone promoter selected from the group consisting of the tryptophan operonpromoter Ptrp of E. coli, a lactose operon promoter Plac of E. coli, aPtac promoter of E. coli, a phage lambda right promoter PR, a phagelambda left promoter PL, a T7 promoter, a promoter of the AOX1 gene fromPichia pastoris, and a promoter of the GAP gene from Pichia pastoris, oris at least one strong promoter selected from the group consisting ofComamonas, Corynebacterium, Brevibacterium, Rhodococcus, Azotobacter,Citrobacter, Enterobacter, Clostridium, Klebsiella, Salmonella,Lactobacillus, Aspergillus, Saccharomyces, Pichia, Zygosaccharomyces,Kluyveromyces, Candida, Hansenula, Dunaliella, Debaryomyces, Mucor,Torulopsis, Methylobacteria, Bacillus, Escherichia, Pseudomonas,Rhizobium, and Streptomyces, and b) at least one ribosome binding sitefrom a phage lambda CII gene or selected from the group consisting ofribosome binding sites from a gene of Comamonas, Corynebacterium,Brevibacterium, Rhodococcus, Azotobacter, Citrobacter, Enterobacter,Clostridium, Klebsiella, Salmonella, Lactobacillus, Aspergillus,Saccharomyces, Zygosaccharomyces, Pichia, Kluyveromyces, Candida,Hansenula, Dunaliella, Debaryomyces, Mucor, Torulopsis, Methylobacteria,Bacillus, Escherichia, Pseudomonas, Rhizobium, and Streptomyces.
 19. Thetransformed microorganism of claim 18, wherein the host microorganism isselected from the group consisting of Comamonas, Corynebacterium,Brevibacterium, Rhodococcus, Azotobacter, Citrobacter, Enterobacter,Clostridium, Klebsiella, Salmonella, Lactobacillus, Aspergillus,Saccharomyces, Zygosaccharomyces, Pichia, Kluyveromyces, Candida,Hansenula, Dunaliella, Debaryomyces, Mucor, Torulopsis, Methylobacteria,Bacillus, Escherichia, Pseudomonas, Rhizobium, and Streptomyces.
 20. Atransformed microorganism selected from the group consisting of: (a) E.coli SW91 having the designation ATCC PTA-1175; (b) E. coli DH5α: pnit4having the designation ATCC PTA-1176; (c) E. coli SS1001 having thedesignation ATCC PTA-1177; and (d) E. coli SS1002 containing plasmidpnitex2; and (e) E. coli SS1011 containing plasmid pnitex2.
 21. A methodof obtaining a nucleic acid fragment encoding all or a substantialportion of a nitrilase enzyme, the method comprising: (a) probing agenomic library with all or a portion of a nucleic acid fragmentselected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ IDNO:3, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9,SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NIO:13, SEQ ID NO:15,and SEQ ID NO:16; (b) identifying a DNA clone that hybridizes with thenucleic acid fragment of step (a); and (c) sequencing the nucleic acidfragment that comprises the DNA clone identified in step (b), whereinthe sequenced nucleic acid fragment of step (b) encodes all or asubstantial portion of an amino acid sequence encoding a nitrilaseenzyme.
 22. A method of obtaining a nucleic acid fragment encoding allor a substantial portion of a nitrilase enzyme, the method comprising:(a) synthesizing at least one oligonucleotide primer corresponding to aportion of the sequence selected from the group consisting of SEQ IDNO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:7,SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQID NO:13, SEQ ID NO:15, and SEQ ID NO:16; and (b) amplifying an insertpresent in a cloning vector using the oligonucleotide primer of step(a), the amplified insert of step (b) encoding all or a substantialportion of an amino acid sequence encoding a nitrilase enzyme.
 23. Theproduct of the method of claims 21 or
 22. 24. A method to enzymaticallyconvert nitrile-containing substrates to a carboxylic acid, the methodcomprising: (a) contacting, under suitable conditions, a transformedheterologous host expressing the polypeptide of claim 4 with anitrile-containing substrate; and (b) optionally collecting thecarboxylic acid produced in step (a).
 25. The method of claim 24,wherein the nitrile-containing substrate is a dinitrile of the formulaNC—R—CN where R is an alkylene group having from 1 to 10 carbon atoms.26. The method of claim 25, wherein the nitrile-containing substrate is2-methylglutaronitrile.
 27. A method to enzymatically convertnitrile-containing substrate(s) to carboxylic acid(s), the methodcomprising: (a) contacting, under suitable conditions, a transformedheterologous host comprising the chimeric gene of claim 9 withnitrile-containing substrate(s); and (b) optionally collecting thecarboxylic acid produced in step (a).
 28. The method of claim 27,wherein the nitrile-containing substrate is a dinitrile of the formulaNC—R—CN where R is an alkylene group having from 1 to 10 carbon atoms.29. The method of claim 27, wherein the nitrile-containing substrate is2-methylglutaronitrile.
 30. The method of claim 27 wherein the suitableregulatory sequences of the chimeric gene comprise an induciblepromoter.
 31. The method of claim 30, the suitable conditions of step(a) further comprising the presence of an inducer of the induciblepromoter.
 32. The method of enzymatically converting2-methylglutaronitrile to the corresponding carboxylic acid, the methodcomprising: (a) contacting, under suitable conditions, E. coli SW91designated ATCC PTA-1175 with 2-methylglutaronitrile; and (b) optionallycollecting the carboxylic acid produced in step (a).
 33. An improvementto the process for preparing five-membered ring lactams or six-memberedring lactams from aliphatic α,ω-dinitriles comprising: (a) contacting analiphatic α,ω-dinitrile in an aqueous reaction mixture with an enzymecatalyst, whereby the aliphatic α,ω-dinitrile is converted to anω-cyanocarboxylic acid ammonium salt; (b) contacting the aqueous productmixture resulting from step (a) with hydrogen and a hydrogenationcatalyst, whereby the ω-cyanocarboxylic acid ammonium salt is converteddirectly to the corresponding lactam without isolation of theintermediate ω-cyanocarboxylic acid, ω-cyanocarboxylic acid ammoniumsalt, ω-aminocarboxylic acid, or ω-aminocarboxylic acid ammonium salt;and (c) recovering the lactam from the aqueous product mixture resultingfrom step (b), the improvement comprising in step (a) contacting analiphatic α,ω-dinitrile in an aqueous reaction mixture with an enzymecatalyst selected from the group consisting of (1) E. coli SW91 havingthe designation ATCC PTA-1175; (2) E. coli DH5a: pnit4 having thedesignation ATCC PTA-1176; (3) E. coli SS1001 having the designationATCC PTA-1177; and (4) E. coli SS1002 containing plasmid pnitex2, and(5) E. coli SS1001 containing plasmid pnitex2.
 34. The process of claim33 wherein the aliphatic α,ω-dinitrile has the formulaNCCX_(a)(R)(CH₂)_(n)CN, where a=0 or 1, where X=hydrogen when a=1, andR═H, alkyl or substituted alkyl, or alkenyl or substituted alkenyl, oralkylidene or substituted alkylidene, and where n=1 or
 2. 35. The methodof claim 34 wherein the α,ω-dinitrile is 2-methylglutaronitrile.
 36. Theprocess of claim 33 wherein the aliphatic α,ω-cyano is unsymmetricallysubstituted at the α-carbon atom, and the enzyme catalyst ischaracterized by aliphatic nitrilase activity that produces theω-cyanocarboxylic acid ammonium salt resulting from regioselectivehydrolysis of the ω-cyano group, thereby producing only one of the twopossible lactam products during step (b).
 37. The process of claim 33further comprising adding, before step (b), ammonium hydroxide, ammoniagas, or methylamine to the aqueous product mixture containing theω-cyanocarboxylic acid ammonium salt
 38. The process of claim 37 whereinthe amount of ammonium hydroxide, ammonia gas, or methylamine added tothe aqueous product mixture is from 0 to 4 molar equivalents relative tothe amount of ω-cyanocarboxylic acid ammonium salt present.
 39. Theprocess of claim 38 wherein methylamine is added to the aqueous productmixture containing the ω-cyanocarboxylic acid ammonium salt before step(b) and the product of step (b) is an N-methyllactam.
 40. The process ofclaim 33 wherein in step (b) the temperature of the aqueous productmixture is from 45° C. to 200° C.
 41. The process of claim 33 forpreparing five-membered ring lactams or six-membered ring lactams fromaliphatic α,ω-dinitriles, wherein: the aliphatic α,ω-dinitrile is ofeither formula:

where R₁ and R₂ are both H, and R₃, R₄, R_(5,) and R₆ are eachindependently selected from the group consisting of H, alkyl orsubstituted alkyl, or alkenyl or substituted alkenyl, or R₃ and R₄ takentogether are alkylidene or substituted alkylidene, or independently R₅and R₆ taken together are alkylidene or substituted alkylidene.
 42. Themethod of claim 33 or 41 wherein the enzyme catalyst is in the form ofwhole microbial cells immobilized in or on an insoluble support.
 43. Amethod for using a native microbial gene encoding a proteincharacterized by a nitrilase activity on nitrilase-containing substratesto obtain a mutated microbial gene encoding a protein characterized byan increased specific nitrilase activity on nitrile-containingsubstrates and/or an increased stability of the nitrilase, one or bothcharacteristics increased relative to that of the native microbial gene,the method comprising the steps of (i) contacting restrictionendonucleases with a mixture of nucleotide sequences to yield a mixtureof restriction fragments, the mixture of nucleotide sequences comprisinga) a native microbial gene; b) a first population of nucleotidefragments which will hybridize with the nucleotide sequences of thenative microbial gene of (i)(a); and c) a second population ofnucleotide fragments which will not hybridize to the nucleotidesequences of the native microbial gene of (i)(a), (ii) denaturing themixture of restriction fragments of step (i); (iii) incubating thedenatured mixture of restriction fragments of step (ii) with apolymerase; and (iv) repeating steps (i), (ii), and (iii) a sufficientnumber of times to yield a mutated microbial gene encoding a proteincharacterized by an increased specific nitrilase activity onnitrile-containing substrates and/or an increased stability of thenitrilase, one or both characteristics increased relative to thenitrilase activity of the native microbial gene.
 44. The method of claim43 wherein the native microbial gene is Acidovorax facilis 72W and thenitrile-containing substrate is 2-methylglutaronitrile.
 45. A mutatedmicrobial gene encoding a protein characterized by an increased specificnitrilase activity on nitrile-containing substrates and/or an increasedstability of the nitrilase, one or both characteristics increasedrelative to the nitrilase activity of a native microbial gene, themutated microbial gene produced by the method of claim
 43. 46. Thetransformed microorganism of claim 19, wherein the host microorganism isE. coli strains MG1655 (ATCC 47076), W3110 (ATCC 27325), MC4100 (ATCC35695), or W1485 (ATCC 12435).